CN103703372B - Anti-human acceptor type Protein-tyrosine-phosphatase σ antibody - Google Patents
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Abstract
With reference to the monoclonal antibody of people's acceptor type protein tyrosine phosphatase σ (people PTPRS) extracellular domain or the fragment including its antigen binding domain.
Description
Technical field
The present invention relates to the antibody for combining people's acceptor type protein tyrosine phosphatase σ.Hereinafter, " protein-tyrosine
Phosphatase " is abbreviated as PTP, and " acceptor type Protein-tyrosine-phosphatase " is abbreviated as RPTP or PTPR, " acceptor type albumen junket ammonia
Acid phosphoric acid enzyme σ " is sometimes referred to as RPTP- σ, PTP- σ or PTPRS, and " people " and " mouse " is represented with prefix h and m respectively sometimes.
Background technology
Interferon (hereinafter, " interferon " is sometimes referred to as IFN) is most important cell in Immune responses of the antivirus
The factor.Interferon generation property cell (IPC in human blood：IPC is undifferentiated lymphatic dendritic cells, and it is positioned
For dendritic cells (DC) precursor.IPC is otherwise referred to as plasmacytoid dendritic cells or plasmacytoid dendritic cells (are starched thin
Born of the same parents' sample dendritic cells：pDC).Hereinafter, IPC and pDC is considered to have identical implication herein, and hereinafter
General rule is standardized as by term pDC) MHC II proteinoid is expressed together with CD4.However, by
In the number of such cell, few and cell causes rapidly Apoptosis and lacks lineage markers, therefore such cell is so far
Not separated or detailed characterizations.It is CD4+CD11c-2- type dendritic cell precursor cells to be proved pDC, and by microorganism
Its caused IFN is 200 to 1000 times caused by other blood cells after stimulation.Therefore, pDC2 is antiviral and antitumor exempted from
Decisive immune system effector cell in epidemic disease response.
IFN α and IFN β are known as the I types IFN with antiviral activity or antitumor activity.On the other hand, clarified
IFN α is related to autoimmune disease.For example, the abnormal production of the IFN α reported in the patient with following autoimmune disease
It is raw.It shows furthermore that mitigate the possibility of the autoimmunity patient's condition by neutralizing IFN α.
Systemic lupus erythematosus (Shiozawa et al., Arthr.＆Rheum.35,412,1992) and chronic class wind are reported
Wet arthritis (Hopkins et al., Clin.Exp.Immunol73,88,1988), and the wherein disease of autoimmune disease
Example that condition is expressed or deteriorated by administered recombinant IFN α 2 or IFN (Wada et al., Am.J.Gastroenterol.90,
136,1995；Perez et al., Am.J.Hematol.49,365,1995；Wilson LE et al., Semin Arthritis,
Rheum.32,163-173,2002)。
In addition, the differentiation of IFN α inducing dendritic cell (DC) is also illustrated.Because dendritic cells and antigen presentation are thin
Born of the same parents, it is taken as that the induction of dendritic cell differentiation constitutes the important mechanisms of autoimmune disease.In fact, it has been proposed that IFN α
Breaking-out closely related (Blanco et al., Science, 16 of induction and systemic lupus erythematosus to dendritic cell differentiation：294,
1540-1543,2001).Therefore, the antitumor activity of IFN α and its substantial connection with autoimmune disease have been had been pointed out.This
Outside, IFN α is also closely related with the breaking-out of psoriasis (Nestle FO et al., J.Exp.Med.202,135-143,2005).
Only a small amount of pDC is present in blood.It is believed that pDC ratio is 1% or less in PBLC.
However, pDC has high generation IFN ability.The ability that pDC produces IFN reaches for example, 3000pg/mL/104Cell.
I.e., it is possible to think that although the number of cell is few, the generation of most of IFN α or IFN β when virus infects in blood is by pDC bands
Come.
PDC is divided into dendritic cells by virus stimulation, and IFN-γ and IL-10 are produced with inducing T cell.In addition, pDC is also
Dendritic cells are divided into by IL-3 stimulation.The dendritic cells inducing T cell broken up by IL-3 stimulation produces Th2 cells
The factor (IL-4, IL-5, IL-10).Therefore, pDC has following characteristics：It depends on the difference stimulated and to be divided into dendron thin
Born of the same parents.
Therefore, pDC is the cell with two aspects：One be as IFN produce property cell aspect, another be make
For the aspect of the precursor of dendritic cells.Two kinds of cells all play an important role in immune system.That is, pDC is never Tongfang
Support one of important cells of immune system in face.
In order to control humoral factor such as IFN activity, the administration for identifying the antibody of the factor is effective.For example,
Be put to using the trial of anti-IL-8 (IL) -1 or IL-4 Antybody therapy autoimmune disease and put into practice (Guler et al.,
Arthritis Rheum,44,S307,2001).In addition, it is believed that the antibody neutralized can turn into for also involving interferon
(IFN) medicine (Stewart, TA.Cytokine Growth the Factor Rev.14 of autoimmune disease；139-
154,2003).Expectable similar method is effective for the IFN as caused by pDC.However, such method is based on producing
Humoral factor effect suppression.If can direct control targe (objective) humoral factor generation, can obtain more
The effect of crucial.
Identification people pDC antibody is reported.For example, anti-BDCA-2 monoclonal antibodies are to be specific to people pDC monoclonal
Antibody (Dzionek A. et al., J.Immunol165：6037-6046,2000).Anti- BDCA-2 monoclonal antibodies are illustrated
(J.Exp.Med.194 is acted on caused by IFN with suppression people pDC：1823-1834,2001).In addition, it there has been reported identification
The monoclonal antibody of mouse interferon generation property cell suppresses interferon and produces (Blood2004Jun1；103/11：4201-
4206.2003 electronic edition in December in year).The monoclonal antibody for having reported anti-mouse pDC reduces the quantity of dendritic cells
(J.Immunol.2003,171：6466-6477).
Provided that similarly identifying people pDC and can control its active antibody, then it is useful.For example, this hair
Bright inventor has illustrated identification Ly49Q antibody specificity combination mouse pDC.However, anti-Ly49Q antibody not interference mice
PDC activity (Blood, 1April2005, volume 105, No.7, pp.2787-2792：WO2004/13325A1).
Phosphoprotein phosphatase is the dephosphorylase found in the research of glycogen metabolism.Except Protein-tyrosine-phosphatase
(PTP) beyond, protein serine/Threonine Phosphatases, phosphatide specificity phosphatase etc. have been observed that these enzymes form egg
The superfamily of white phosphatase.Among these enzymes, Protein-tyrosine-phosphatase is observed in the tyrosine residue in protein
It is responsible for the enzyme of phosphorylation among the reversible phosphorylation modification arrived.In another aspect, protein tyrosine kinase (PTK) is by example
Reversible phosphorylation to be observed in the tyrosine residue in protein is responsible for the enzyme of phosphorylation in modifying.
Ligand binding information in its extracellular domain is changed into intracellular structure by Protein-tyrosine-phosphatase (PTP)
The phosphatase activity in domain, it is believed that protein tyrosine kinase (PTK) is activated by the combination of part, and Protein-tyrosine-phosphatase
(PTP) generally inactivated by the combination of part.Therefore, in Protein-tyrosine-phosphatase (PTP) and protein tyrosine kinase (PTK)
In, the stimulation of part results in the rise of phosphorylation level, and expected in the presence of very big difference in signal properties.In albumen junket
In the case of histidine kinase (PTK), the wherein acceptor positive feedback of phosphorylation and activation control, and albumen junket each other has been carried out
The Local activation of histidine kinase (PTK) molecule is transferred to other protein tyrosine kinases (PTK) molecule on cell membrane, so as to phosphorus
Acidifying increases in a wide range.On the other hand, the only bound molecule of part is gone out in Protein-tyrosine-phosphatase (PTP)
It is living, and the phosphorylation of substrate only locally increases.Participate in the Protein-tyrosine-phosphatase of many physiological functions and cell function
(PTP) (Division is largely paid close attention in the wide spectrum of cranial nerve biochemistry, immunology, cancer, diabetes etc.
The copy of of Molecular Neurobiology, National Institute for Basic Biology homepage,
http://niwww3.nibb.ac.jp/RPTP.pdf)。
Protein-tyrosine-phosphatase family can be classified into acceptor type of the cell membrane through area and non-acceptor type.
The molecule of 21 kinds of acceptor type Protein-tyrosine-phosphatases (being also abbreviated by RPTP or PTPR) in mammal be present, it is divided
Into 8 subfamilies, each subfamily has intrinsic extracellular structure, wherein observing immunoglobulin like domain, fibre
Company's protein types III spline structures domain, carbonate dehydratase spline structure domain, MAM domains etc. (Nat Rev Mol Cell Biol,
Volume 7,833-846,2006).
(this is abbreviated as hRPTP- σ, hPTP- σ or hPTPRS to people's acceptor type protein tyrosine phosphatase σ, main herein
To use abbreviation hPTPRS) and LAR (LAR) and acceptor type protein-tyrosine phosphorus
Sour enzyme δ (PTP- δ) belongs to R2A subfamilies together.The enzyme of PTPR families is in different tissue (including since the generation of animal
(initiation of generation) is to the nervous system after ripe) in express, but its seldom physiological function is elucidated with,
Because the identification of ligand molecular and substrate molecule is not easy.
Dendritic cells (DC) are the major antigen presenting cells in live body, and it is present in blood, lymphoid tissue etc., and
By rude classification pulpefaction sample dendritic cells (mDC) and plasmacytoid dendritic cells (pDC).PDC selectivity on its cell surface
TLR7 and TLR9 is expressed as Toll-like receptor, and produces I types interferon-' alpha ' and β, especially interferon-' alpha '.
Nearest research, which has illustrated, acts on dendritic cells to control the various ligand moleculars of their maturation and activation, and
Cellular Signaling Transduction Mediated mechanism (intracellular signal transmission mechanism) from its acceptor is
It is made apparent from.However, also there are many unclear parts in the mechanism on modification and the control of the function of dendritic cells.With many
Illustrating in other cells is similar, and the phosphorylation of protein is considered as also controlling the signal for carrying out autoreceptor to pass in dendritic cells
Lead in motion/migration of (signal transmission), cell etc. and play an important role.
Phosphoprotein phosphatase as the negative control factor of protein phosphorylation is as maintaining to adjust dendritic cells
The dominant candidate person of the factor of the appropriate intensity and length of the signal of activation and function.(Nobuhiro Tanuma(Institute
for Genetic Medicine,Hokkaido University),Northern Advancement Center for
Science＆Technology (abbreviations：NOASTEC in homepage) " tyrosine phosphatase induced in dendritic cell maturation
Functional analysis ", http://ww.noastec.jp/kinouindex/data2005/pdf/01/01_20.pdf).
International publication No.W095/9656A1 discloses RPTP- σ (PTPRS) and encodes its nucleic acid；However, disclosed ammonia
Base acid sequence is derived from the amino acid sequence of rat, and the announcement does not refer to the antibody for being specific to PTPRS.International publication
No.W095/9656A1 is also without open Anti-Human's PTPRS antibody.
International publication No.WO2007/41317A1 is related to specific binding at least a kind of RPTP- σ or RPTP- δ (to suppress
The immune response of immunocyte) or its antigen-binding fragment separation antibody.Document is described by using specific binding
RPTP antibody, poxvirus polypeptide A 41L and RPTP combination are by Reverse transcriptase, it is achieved thereby that the immune of immunocyte should
The suppression answered.However, the document does not disclose the antibody that actually obtained for specifically binding RPTP- σ (PTPRS), just consider
For the description of embodiment, embodiment only confirms that the combination A41L expressed in immunocyte RPTP is to belong to identical
RPTP- σ, the RPTP- δ of R2A hypotypes and a LAR part and it is prepared for LAR (LAR (Ig domains)-Fc merges egg with Fc
The fusion protein of immunoglobulin like domain in vain).Be difficult to international publication No.WO2007/41317A1 disclose it is only special
Antibody in RPTP- σ and the preparation to it.
Only in conjunction with RPTP- σ (specific site of the PTPRS i.e. in the application) antibody and RPTP- σ can be specifically bound
(PTPRS) antibody for but not combining the RPTP- δ and LAR that belong to identical R2A hypotypes does not obtain also.People PTPRS is it in pDC
The molecule that is observed of expression, but any anti-human PTPRS antibody does not obtain yet until now.
Quotation list
Patent document
PTL1：WO2004/13325A1
PTL2：W095/9656A1
PTL3：WO2007/41317A1
Non-patent literature
NPL1Shiozawa et al., Arthr.＆Rheum.35,412,1992
NPL2.Hopkins et al., Clin.Exp.Immunol.73,88,1988
NPL3.Wada et al., Am.J.Gastroenterol.90,136,1995
NPL4Perez et al., Am.J.Hematol.49,365,1995
NPL5Wilson LE et al,Semin Arthritis.Rheum.32,163-173,2002
NPL6Blanco et al., Science, 16:294,1540-1543,2001
NPL7Nestle FO et al., J.Exp.Med.202,135-143,2005
NPL8Guler et al., Arthritis Rheum., 44.S307,2001
NPL9Stewart,TA.Cytokine Growth Factor Rev.14；139-154,2003
NPL10：Dzionek, A. et al. J.Immunol.165：6037-6046,2000
NPL11：J.Exp.Med.194：1823-1834,2001
NPL12：Blood2004Jun1;103/11:4201-4206.Epub2003Dec
NPL13：J.Immunol.2003,171:6466-6477
NPL14：Blood,1April2005,Vol.105,No.7,pp.2787-2792
NPL15：http://niwww3.nibb.ac.jp/RPTP.pdf
NPL16：Nat Rev Mol Cell Biol.,Vol.7,833-846,2006
NPL17：
http://www.noastec.jp/kinouindex/data2005/pdf/01/01_20.pdf
Summary of the invention
Technical problem
It is an object of the invention to provide combine people's acceptor type protein tyrosine phosphatase σ's (people PTPRS, hRPTP- σ)
Antibody and detection, identification or separation pDC.In addition, the purpose of the present invention is to adjust pDC activity.
The present inventor confirms that expression of the PTPRS in pDC is increased by specificity by the research related to people pDC
By force.Therefore, the present invention attempts to prepare PTPRS antibody and illustrates its effect.
In order to obtain the antibody of the protein from live body of identification trace, it will generally utilize and pass through gene recombination technology
The protein of preparation is used as immunogene.The present inventor attempted the cDNA sequence based on the people PTPRS being elucidated with
And the information table intelligent PTPRS (GenBank accession number NM_002856.3) of the amino acid sequence on being encoded by it.
In order to obtain the antibody of protein, generally attempt to the partial amino-acid series of native protein being used as immunogene.
However, in order that antibody identification cell surface on molecule, such region should be selected：It is formed is identified as cell by antibody
The part of epitope on surface.It is therefore contemplated that people is specific to obtain as immunogene by using fragment amino acid sequence
PTPRS antibody or remote.
The solution of problem
It that case, the antibody that the present inventor has illustrated with reference to pDC can be by using special immune
Originally obtain.In addition, they also have confirmed that obtained antibody specificity identification people pDC and with its active works of regulation
With, and complete the present invention.
Therefore, the present invention relates to following Anti-Human PTPRS antibody, for producing the method and its application of the antibody.
The present invention is as follows.
(1) monoclonal antibody of people's acceptor type protein tyrosine phosphatase σ (people PTPRS) extracellular domain is combined
Or the fragment including its antigen binding domain.
(2) according to the monoclonal antibody of above-mentioned (1) or the fragment including its antigen binding domain, it combines plasmacytoid dendritic
Cell.
(3) by with the hybridoma 9H5-4 of accession number FERM BP-11356 preservations, with accession number FERM BP-11357 preservations
Hybridoma 10F7-38, with the hybridoma 13G5-52 of accession number FERM BP-11358 preservations, with accession number FERM BP-
The hybridoma 13G5-57 of 11359 preservations, with the hybridoma 14A8-85 of accession number FERM BP-11360 preservations, with accession number
The hybridoma 22H8-84 of FERM BP-11361 preservations, with the hybridoma 49F2-30 of accession number FERM BP-11362 preservations, with
Monoclonal antibody or the piece including its antigen binding domain caused by the hybridoma 55E7-79 of accession number FERM BP-11363 preservations
Section.
(4) produce according to above-mentioned (1) or the hybridoma of any monoclonal antibody of (2).
(5) by with the hybridoma 9H5-4 of accession number FERM BP-11356 preservations, with accession number FERM BP-11357 preservations
Hybridoma 10F7-38, with the hybridoma 13G5-52 of accession number FERM BP-11358 preservations, with accession number FERM BP-
The hybridoma 13G5-57 of 11359 preservations, with the hybridoma 14A8-85 of accession number FERM BP-11360 preservations, with accession number
The hybridoma 22H8-84 of FERM BP-11361 preservations, with accession number FERM BP-11362 preservations or hybridoma 49F2-30 or
With monoclonal antibody caused by the hybridoma 55E7-79 of accession number FERM BP-11363 preservations or including its antigen binding domain
Fragment.
(6) method for being used to produce monoclonal antibody, it includes culture and received according to the hybridoma of above-mentioned (5) and from culture
Collect monoclonal antibody.
(7) it is used to produce celliferous method, the cell produces the monoclonal antibody with reference to people PTPRS, methods described bag
Include：
1) animal for treating immunity inoculation is given to include the thin of the exogenous proteins of people PTPRS extracellular domain using expression
Born of the same parents, and
2) the antibody production of the antibody with reference to people PTPRS is produced from the antibody-producing cell selection of the animal through immunity inoculation
Natural disposition cell.
(8) according to the method for above-mentioned (7), wherein expression people PTPRS cell is to keep coding to include people PTPRS with expressing
Extracellular domain amino acid sequence exogenous polynucleotide cell.
(9) according to the method for above-mentioned (8), wherein cell is zooblast.
(10) according to the method for above-mentioned (9), wherein cell is the cell in mouse source.
(11) according to the method for above-mentioned (10), the cell in wherein mouse source is D2SC cells.
(12) according to the method for any one of above-mentioned (7) to (11), it is also obtained antibody producing thin including clone
Born of the same parents.
(13) method for being used to produce the monoclonal antibody of the extracellular domain with reference to people PTPRS, methods described include
The antibody-producing cell by being obtained according to the method for above-mentioned (9) is cultivated, and monoclonal antibody is collected from culture.
(14) people PTPRS monoclonal antibody or the fragment including its antigen binding domain are identified, the antibody can be under
Row step obtains：
1) protein of the extracellular domain including people PTPRS is expressed to the animal for treating immunity inoculation with applying external source
Cell；
2) the antibody production of the antibody with reference to people PTPRS is produced from the antibody-producing cell selection of the animal through immunity inoculation
Natural disposition cell；With
3) antibody-producing cell of selection in (2) is cultivated, and identification people PTPRS antibody is collected from culture.
(15) (a) is used for the immunogene for producing the antibody with reference to people PTPRS, and it is with including external source and expression ground keeps coding
The zooblast of the polynucleotides of the amino acid sequence of extracellular domain including people PTPRS, or its cellular membrane fractions.
(16) according to the immunogene of above-mentioned (15), wherein zooblast is the cell of people source.
(17) method for being used to detect plasmacytoid dendritic cells, it includes that people PTPRS extracellular domain will be combined
Monoclonal antibody or fragment including its antigen binding domain contacted with subject cell, and detection resists with reference to the monoclonal of cell
Body, or the fragment including its antigen binding domain.
(18) it is used for the reagent for detecting plasmacytoid dendritic cells, it includes the extracellular domain with reference to people PTPRS
Monoclonal antibody, or the fragment including its antigen binding domain.
(19) it is used for the active method for suppressing plasmacytoid dendritic cells, it is included any of following component and slurry
Cell sample dendritic cells contact：
(a) people PTPRS is combined to suppress the active monoclonal antibody of plasmacytoid dendritic cells or including its antigen knot
The fragment in area is closed, and
(b) immunoglobulin of the complementary determining region of the monoclonal antibody of (a) has been transplanted to it, or including its antigen knot
Close the fragment in area.
(20) it is used for the active method for suppressing the plasmacytoid dendritic cells in live body, it includes applying arbitrarily to live body
Following component：
(a) people PTPRS is combined to suppress the active monoclonal antibody of plasmacytoid dendritic cells or including its antigen knot
The fragment in area is closed, and
(b) immunoglobulin of the complementary determining region of the monoclonal antibody of (a) has been transplanted to it, or including its antigen knot
Close the fragment in area.
(21) according to the method for above-mentioned (19) or (20), the activity of wherein plasmacytoid dendritic cells is interferon generation property
One or both of survival of activity and interferon generation property cell.
(22) be used to suppress the active reagents of plasmacytoid dendritic cells, it include any following component as it is active into
Point：
(a) people PTPRS is combined to suppress the active monoclonal antibody of plasmacytoid dendritic cells or including its antigen knot
The fragment in area is closed,
(b) immunoglobulin of the complementary determining region of the monoclonal antibody of (a) has been transplanted to it, or including its antigen knot
Close the fragment in area.
(23) it is used to suppress the active reagent according to the interferon of above-mentioned (22) generation property cell, wherein Plasmacytoid tree
The activity of prominent cell is one or both of survival of interferon generation property activity and interferon generation property cell.
The favourable effect of the present invention
Exempt from the invention provides specific recognition people PTPRS (immunogene for being used to produce antibody) antibody and for utilizing
The method that epidemic focus produces Anti-Human's PTPRS antibody.People PTPRS is the memebrane protein for belonging to RPTP families.The present inventor illustrates
Specific recognition people PTPRS antibody can be readily attained.Can be tool by Anti-Human's PTPRS antibody that the present invention obtains
There is the antibody of high degree of specificity, it distinguishes people pDC with expressing the cell of other RPTP families.
In preferred embodiments, Anti-Human PTPRS antibody binding people pDC provided by the invention.In addition, the antibody of invention
Specific recognition people pDC.Therefore, it can be used for detecting and separating pDC.PDC is the cell for producing most of 1 type IFN.Therefore,
It is detected and separation involves pDC disease wherein（Such as autoimmune disease）Diagnosis and research in be important.
In addition, Anti-Human PTPRS antibody provided by the invention has in preferred embodiments adjusts the active of people pDC
Effect.Therefore, Anti-Human PTPRS antibody of the invention can be used for the activity for suppressing pDC.Therefore, if using antibody of the invention
Suppress pDC activity, then in the patient for the autoimmune disease that the expression with wherein IFN α has strengthened, be contemplated that
Therapeutic effect.
PDC produces a large amount of IFN by few cell.In order to neutralize IFN, the antibody corresponding to IFN molecular number is must
Need.However, in the present invention, the activity of caused cell is inhibited directly.Therefore, compared to by anti-IFN antibody
With utilize the expectable effect for producing stronger suppression IFN of lesser amount of antibody.In addition, it ought persistently produce IFN situation
Under, it is contemplated that neutralization of the antibody to IFN is only instantaneously suppressed, but pDC activity inhibited, so that expectable length in the present invention
Effect caused by the suppression IFN of phase.
Brief description of the drawings
Fig. 1 is PTPRS amino acid sequence (SEQ ID NO:1).PTPRS is that have immune globulin in extracellular space
The single membrane span memebrane protein in white spline structure domain (Ig- spline structures domain) and fi-bronectin type III spline structure domain.In addition, it is in the cell
There are two Protein-tyrosine-phosphatase regions (PTP fabric domain) in region；
Fig. 2 is the figure for showing PTPRS relative expression levels in different immunocytes.Show PTPRS with pDC specificity
Mode is expressed；
Fig. 3 is the figure for showing the comparison of the expression of PTPRS genes between tissue.PTPRS mRNA are shown in spleen and ovary
Relatively high expression, and also widely expressed in other tissues；
Fig. 4 shows the selection of the expression people PTPRS (hPTPRS) carried out by FACS sortings cell；
The FACS screenings for the hybridoma that Fig. 5 displays are carried out using the hPTPRS/D2SC/1 cells through immunity inoculation.Obtain
13 hybridomas for producing anti-hPTPRS antibody；
Fig. 6 shows the FACS screenings carried out using CAL-1 cells；
Fig. 7 shows the FACS screenings carried out using human peripheral pDC；
Fig. 8 is the figure for the homology for showing hPTPRS and other PTPR.PTPRS belongs to PTPR families, this family it is several
The amino acid sequence of family molecule and PTPRS amino acid sequence have high homology；
Fig. 9 is the Hybridoma Cell Culture for the antibody for identifying and producing specific binding people pDC for showing 10 types
Whether supernatant (2G6,4B2,2G2,9H5,10F7,22H8,49F2,14A8,55E7,13G5) only specifically binds PTPRS
The test result of (hPTPRE is not expressed on cell surface).As a result, 2G6 is shown and PTPRF cross reactivity (figure
9, D), 4B2 is shown and PTPRD cross reactivity (Fig. 9, C).The antibody of other 9 types shows PTPRS specific bindings
(Fig. 9, A to D)；
Figure 10 is the test result of anti-PTPRS antibody and the cross reactivity of monkey.All Hybridoma Cell Culture supernatants
Liquid specifically binds the pDC cell masses (pedigree-CD123+HLA-DR+) of machin；
Figure 11 shows individualized (singlization) sorting of hybridoma.Unicellular sorting is by using FACS Aria
(BD) carry out, D2SC/1 cells and hPTPRS/D2SC/1 cells (A and B), CAL-1 cells (C) and people pDC (D) be by making
With the cell culture supernatant of hybridoma come what is dyed, single hybridoma is selected；
Figure 12 shows the endotoxic concentration in the antibody of the purifying of the culture supernatant obtained from hybridoma.All concentration are
Standard value 0.3Eu/mg Ab or smaller；
Figure 13 is the test result of the ability of the people PTPRS on the antibody binding cell surface of purifying.It can confirm all anti-
Body maintains their binding ability；
The specific binding of the pDC cell masses (BDCA2+) of the antibody on human peripheral blood of Figure 14 display purifying；
Figure 15 be test Anti-Human PTPRS antibody whether also in relation with mouse PTPRS test result.49F2-30、13G5-
52nd, 13G5-57 and 22H8-84 are bound to mPTPRS/CHO；
Figure 16 shows the complement dependent cytotoxicity activity of the anti-PTPRS antibody of anti-hPTPRS expressivities cell.Measurement is anti-
The complement dependent cytotoxicity activity of people PTPRS/CHO (Figure 16 A) and mouse PTPRS/CHO (Figure 16 B) anti-PTPRS antibody.
As a result, 13G5-52 and 13G5-57 show that the CDC of about 20% anti-human PTPRS/CHO target is active (A), and 13G5-52
Show that the CDC of about 100% anti-mouse PTPRS/CHO target is active (B) with 13G5-57；
Figure 17 show the ch49F2-30 (Figure 17 A) of anti-hPTPRS chimeric antibodies, ch9H5-4, ch13G5-57 and
Ch22H8-84 (Figure 17 B) damages target hPTPRS/CHO in a manner of effector cell's number dependence；With
Figure 18 shows the generation of IFN α completely by with ch49F2-30, ch9H5-4, ch13G5-57 and ch22H8-84
The processing of anti-PTPRS chimeric antibodies inhibits (Figure 18 A), has illustrated pDC colonies and has subtracted in the Synagis processing of control antibodies
Few more (Figure 18 B and Figure 18 C).
The description of embodiment
People PTPRS is that its specific expressed molecule is observed in plasmacytoid dendritic cells pDC.However, for producing
Any method of raw identification people PTPRS antibody is not set up still.
Known people PTPRS 4 isotypes, its isotype 1 for including being made up of 1948 amino acid residues, by 1910
The isotype 2 of amino acid residue composition, the isotype 3 that is made up of 1501 amino acid residues and by 1505 amino acid residue groups
Into isotype 4.In its structure, it was observed that 3 immune globulin spline structure domain (the first Ig structures as extracellular structure
Domain, the 2nd Ig domains and the 3rd Ig domains), fi-bronectin type III spline structure domain, membrane spaning domain (membrane spaning domain, TM
Region) and two phosphatase domains (D1 and D2 domains) as intracellular structure.Only tied with the close D1 of cell membrane
Structure domain has Protein-tyrosine-phosphatase (PTP) active.In Fig. 1, signal peptide and typical domain are in amino acid sequence
It is labeled to come out.
People PTPRS isotype 3 is to include SEQ ID NO:1 831 to 851 (Fig. 1) wear film as membrane spaning domain
Albumen.In 1501 amino acid residues including N-terminal, 29 amino acid residue (SEQ ID NO:1 to 29 in 1) form
Signal sequence, 30 to 830 form extracellular domain.On the other hand, C-terminal side is intracellular domain.Think extracellular
The activity of part control PTPRS in environment.
The present inventor by gene expression analysis confirm people PTPRS in people pDC it is specific expressed.They
Think, if the antibody that can distinguish people PTPRS and other molecules can be obtained, it can be used for pDC research.However, in the presence of being permitted
The molecules with the similar structure in the PTP families including people PTPRS more.Molecule is, for example, RPTP- σ and PTPRA (RPTP-
α), (RPTP- ε, PTPRF (RPTP- ζ) PTPRS are especially included with high homology by PTPRD (RPTP- δ), PTPRE
Amino acid sequence (Fig. 8).Therefore, they think to be difficult to by using domain peptides, using the amino for forming extracellular domain
The partial sequence of acid sequence obtains the antibody that these molecules can be distinguished from each other as immunogene.Therefore, invention of the invention
People attempts to obtain anti-human PTPRS antibody as the cell of immunogene by using expression people PTPRS.
The present inventor has been carried out research in detail and, to obtain identification people PTPRS antibody, illustrates target and resist
Body can be obtained by using specific convertibility cell as immunogene, and complete the present invention.That is, the present invention relates to combination
The monoclonal antibody of people PTPRS extracellular domain or the fragment including its antigen binding domain.
In the present invention, people PTPRS can be defined as the natural molecule expressed in people pDC, or in immunology with
Molecule equivalent the people PTPRS that is expressed in people pDC.In the present invention, antibody binding people PTPRS can be for example confirmed as follows.
- based on the reactive confirmation with people's cell：
The discovery obtained according to the present inventor, it is believed that people PTPRS can be used as pDC mark, since it is observed that
It is specific to people pDC expression.
Such express spectra based on people PTPRS, first, the activity of pDC combination at least a portion subgroups is in the present invention
One of key character of antibody with reference to people PTPRS.Specific cells are that pDC can be by being intrinsic for each cell mass
Cell surface marker confirm.For example, the combination to target cell is by using the antibody for combining cell surface marker
Double dyeing of antibody to be confirmed confirm with its binding activity.That is, pDC of the invention includes the cell for for example expressing BDCA2.
- reactive the confirmation based on the transformed cells with expressing people's PTPRS genes：
The present inventor has confirmed that, when expressing people's PTPRS genes under given conditions, is reconstructed in people pDC
The people PTPRS of expression amynologic characteristic.Therefore, the reactivity with people PTPRS can be based on anti-by encoding human PTPRS gene
The reactivity of the antibody of cell therein is artificially introduced to confirm.That is, the present invention relates to monoclonal antibody or including its antigen
The fragment of land, the monoclonal antibody combine the amino acid sequence conduct for the extracellular domain for including forming people PTPRS
The molecule of extracellular domain.Meanwhile extracellular domain is by corresponding to SEQ ID NO:The amino acid sequence of the 30 to 830 of 1
(Fig. 1) is (from SEQ ID NO：The N-terminal of the amino acid sequence shown in 1) form.
For example, in the cell converted with the expression vector for the DNA for including encoding human PTPRS, expressed in people pDC
PTPRS amynologic characteristic is maintained.Therefore, people PTPRS transformed cells are expressed preferably as being used to confirm in the present invention
The cell of the binding property of the antibody of anti-human PTPRS extracellular domain.Confirmed when in the present invention by transformed cells
During the reactivity of antibody, it is desirable to be used as control by the use of the cell not being converted.
Then, the antibody in the present invention with reference to people PTPRS can be its PTP family with known expression in addition to people PTPRS
Cell mass the cross reactivity antibody that is observed or is not observed.The antibody that its cross reactivity is not observed is excellent
Elect the antibody that people PTPRS is combined in the present invention as.And specifically, it is preferable to following antibody are as resisting with reference to people PTPRS in the present invention
Body：In the condition with confirming to be combined with pDC under the same conditions, with the known cell mass for expressing the PTP families in addition to people PTPRS
The antibody being combined.
That is, the monoclonal antibody in the present invention with reference to people PTPRS extracellular domain preferably includes have following be immunized
Learn the monoclonal antibody of feature.
A) it combines people pDC,
B) under conditions of it combines people pDC wherein, it is to positive selected from monocyte, macrophage, B cell and CD34
One or more combinations can not be identified in cell and dendritic cells from this kind of cell.
Specifically, wherein under conditions of antibody binding people pDC, its to selected from monocyte, macrophage, B cell and
The antibody that one or more combinations can not be identified in CD34 positive cells and dendritic cells from this kind of cell is preferred
Monoclonal antibody as the present invention.
Or the monoclonal antibody of the extracellular domain in the present invention with reference to people PTPRS preferably includes have following exempt from
The monoclonal antibody of immunological features.
C) it combines the transformed cells for keeping encoding human PTPRS DNA expression vector to convert with expression ground,
D) under conditions of for reference to the transformed cells in c), the combination before the conversion in c) to host cell is not
It can be identified.
In the present invention, Anti-Human PTPRS monoclonal antibodies can not pass through with other molecule cross reactions in PTP families
Using wherein the cell of each PTP family of forced expression confirms.That is, the amino acid sequence of each PTP family is encoded
CDNA be forced to express by being introduced into appropriate host cell.Its cross reactivity Anti-Human PTPRS to be confirmed is mono-
Clonal antibody contacts with the transformed cells obtained.Then, if other PTP families with expression in addition to people PTPRS are not observed
The combination of the cell of race's molecule, then it can confirm that antibody can distinguish people PTPRS and other PTP family molecules in immunology.For example,
In the following embodiments, it was confirmed that by most of Anti-Human PTPRS monoclonal antibodies for obtaining of the present invention not with especially with
PTPRS has PTPRA, PTPRD and PTPRF cross reaction of high homology.Therefore, with reference to people PTPRS and it is identical
Under the conditions of the monoclonal antibody that is not detected at of combination to PTPRA, PTPRD and PTPRF be that the preferred monoclonal of the present invention resists
Body.If using the antibody that can be distinguished this kind of PTP family molecules and PTPRS in immunology, can specifically detect
To the change of PTPRS expression.In addition, it was demonstrated that have with PTPRS among the molecule of high homology, can be in cell
Confirm PTPRE expression, but PTPRE is not in extracellular expression.Therefore, it is not as antibody binding PTPRE.
Combination between its binding activity monoclonal antibody to be confirmed and various cells is using such as flow cytometry
Principle confirm.In order to confirm the reactivity of antibody using the principle of flow cytometry, in advance with generation detectable signal
Molecule or atomic group to carry out labelled antibody be favourable.Normally, using fluorescent marker or luminous marker.In order to utilize stream
Combination between the principle analysis fluorescent labeled antibody and cell of formula cell art, the cell sorter of fluorescence-activation can be used
(FACS).By using FACS, the combination between Multiple Antibodies and cell can be effectively confirmed.
Specifically, for example, will illustrate in advance the antibody A that can identify pDC and its have with reference to pDC property it is to be analyzed
Antibody B and one group of cell simultaneous reactions for including pDC.Advance with the fluorescence signal labelled antibody A and antibody that can be distinguished from each other
B.If detecting two kinds of signals in identical cell mass, those antibody binding identical cell masses can be confirmed.That is, can send out
Existing, antibody A and antibody B have identical binding property.If they combine different cell masses, they apparent combination
Property is different.
The example of the preferred monoclonal antibody of the present invention may include by hybridoma 9H5-4,10F7-38,13G5-52,13G5-
57th, monoclonal antibody caused by 14A8-85,22H8-84,49F2-30 or 55E7-79.
Hybridoma 9H5-4,10F7-38,13G5-52,13G5-57,14A8-85,22H8-84,49F2-30 and 55E7-79
Respectively with accession number FERM BP-11356, FERM BP-11357, FERM BP-11358, FERM BP-11359, FERM BP-
11360th, FERM BP-11361, FERM BP-11362 and FERM BP-11363 were deposited in independent administration on April 1st, 2011
International special permission biological deposits center (the International Patent Organism of legal person's industrial technology comprehensive study institute
Depositary(IPOD),National Institute of Advanced Industrial Science and
Technology(NAIST)).Hereinafter, the content on illustrating preservation will be described.
(a) title of preservation mechanism：The international special permission biological deposits center of Independent Administrative Leged Industrial Technology Complex Inst
(International Patent Organism Depositary(IPOD), National Institute of
Advanced Industrial Science and Technology(NAIST))。
Address：Tsukuba Central 6.1-1-1Higashi, Tsukuba, Ibaraki305-8566, Japan
(b) preservation date：On April 1st, 2011
(c) accession number FERM BP-11356 (hybridoma 9H5-4)
(c) accession number FERM BP-11357 (hybridoma 10F7-38)
(c) accession number FERM BP-11358 (hybridoma 13G5-52)
(c) accession number FERM BP-11359 (hybridoma 13G5-57)
(c) accession number FERM BP-11360 (hybridoma 14A8-85)
(c) accession number FERM BP-11361 (hybridoma 22H8-84)
(c) accession number FERM BP-11362 (hybridoma 49F2-30)
(c) accession number FERM BP-11363 (hybridoma 55E7-79)
The monoclonal antibody of the present invention can include the fragment of its antigen binding domain.E.g., including pass through IgG enzymatic
The antibody fragment of antigen-binding site caused by digestion also is used as the antibody of the present invention.Specifically, antibody fragment such as Fab or
F (ab') 2 can be digested by using papain or pepsin to obtain.It is well known that this kind of antibody fragment can use
Make the antibody molecule with the binding affinity for antigen.Or as long as required antigen-binding activity is maintained, may be used also
Use the antibody built by genetic recombinants.The example of the antibody built by genetic recombination may include that chimeric antibody, CDR move
Plant antibody, scFv, binary (diabody), linear antibodies, multi-specificity antibody for being formed by antibody fragment etc..For based on
The method that the antibody-producing cell of monoclonal antibody or generation monoclonal antibody obtains this antibody-like is known.
Specific transformed cells can be used as immunogene to obtain in the monoclonal antibody of the present invention.That is, the present invention relates to
In producing celliferous method, the cell produces the monoclonal antibody of the extracellular domain with reference to people PTPRS, methods described bag
Include：
(1) exogenous proteins of people PTPRS extracellular domain are included using expression to the animal through immunity inoculation
Cell, and
(2) people PTPRS antibody-producing cell is combined from the antibody-producing cell selection of the animal through immunity inoculation.
, can be from culture by cultivating the antibody-producing cell so obtained or the antibody-producing cell immortalized
Thing collects desired monoclonal antibodies.For the method for immortalized antibody generation property cell, various methods are known.
The transformed cells of immunogene as the present invention can obtain for example, by preparing following cell, the cell table
Exogenous polynucleotide is kept up to ground, its (a) coding includes the amino acid sequence of people PTPRS extracellular domain.
In the present invention, exogenous polynucleotide refers to that polynucleotides are artificially introduced host cell.When people is thin
In the case that born of the same parents are used as cell, people's gene is introduced into people's cell.Also in such combination, the polynucleotides being artificially introduced are claimed
For exogenous polynucleotide.Therefore, people PTPRS ectopic expression is included in the expression of exogenous polynucleotide.
In the present invention, people PTPRS extracellular domain refers to the amino acid sequence from 30 to 830 positions, and its is right
Should be in SEQ ID NO：The extracellular domain of amino acid sequence described in 1.For example, include in order from following N-terminal sides
The amino acid sequence in each region is preferably the amino acid sequence that the present invention includes people PTPRS extracellular domain.
[signal sequence+extracellular domain+membrane spaning domain+intracellular space]
Or partly lack following intracellular space amino acid sequence also covered in the present invention include people PTPRS
In the amino acid sequence of extracellular domain.
[part of signal sequence+extracellular domain+membrane spaning domain+intracellular space]
In addition, the structure for lacking intracellular space as follows is also included within the extracellular structure for including people PTPRS of the present invention
In the amino acid sequence in domain.
[signal sequence+extracellular domain+membrane spaning domain]
In said structure, SEQ ID NO are selected from except the overseas region of extracellular structure can have:Amino acid shown in 1
The sequence of sequence, or combined include other homologous amino acid sequences.For example, form signal sequence, membrane spaning domain and thin
The amino acid sequence of intracellular region can be the amino acid sequence of the PTP family molecules in addition to people PTPRS.
Or the amino acid sequence of the PTP families of the combined species in addition to people.Extracellular domain is removed in addition, forming
The amino acid sequence in outer region may include the mutation of the degree to the function that can maintain each region.In addition, can be by other regions
Insert between each region.For example, epitope tag such as FLAG can be inserted between signal sequence and extracellular domain.Specifically
Ground, signal sequence are the regions for being translated into protein, are processed in the transition phase to cell membrane surface, and be removed.
Therefore, any amino acid sequence for inducing the cell membrane of translated protein to pass through is used as signal sequence.More specifically,
People PTPRS amino acid sequence (SEQ ID NO:1) amino acid sequence of people PTPRS extracellular domain is preferably comprised.
Therefore, for the polynucleotides of above-mentioned (a) in the composition present invention, coding can be used to form said structure [signal
Sequence+extracellular domain+membrane spaning domain+intracellular space] amino acid sequence any base sequence.For example, SEQ
ID NO:1 amino acid sequence is by SEQ ID NO:Base sequence coding described in 2.
In the present invention, in order to obtain the transformed cells of immunogene to be used as, it is only necessary to expression vector as introducing, wherein
It is maintained in appropriate host cell to above-mentioned polynucleotides (a) expression.
Host cell in the present invention is preferably mammalian cell.Specifically, from people, monkey, mouse or rat
Cell can be used as host cell.
Specifically, the cell of people source can be host cell.For example, HEK-293T cells are a kind of Human embryo sources
Kidney cell line, it is used as the host cell in the present invention.HEK-293T cells can be obtained as ATCC CRL-11268.
Other cells from the animal for treating immunity inoculation also are used as host cell.When from the thin of the animal through immunity inoculation
When born of the same parents are used as immunogene, the immune response of anti-host cell is small.Therefore, the people expressed while anti-external source can be effectively obtained
The antibody of PTPRS extracellular domain.Thus, for example, when mouse to be used as to the animal through immunity inoculation, mouse source
Cell may also used as host cell.
Can by can in host cell on the carrier of induced expression loading multi-core thuja acid by above-mentioned polynucleotides turn
Change enters cell.Can be used can in mammalian cell induced expression the carrier being obtained commercially.Expression vector such as pCMV-
Script (R) carrier, pSG5 carriers (by Stratagene productions) and pcDNA3.1 are available (by Invitrogen productions)
In the present invention.
If necessary, can by the transformed cells so obtained together with extra component (such as adjuvant) to through immunity inoculation
Animal is applied.As adjuvant, Freund's complete adjuvant etc. can be used.When the situation that mouse is used as to the animal through immunity inoculation
Under, can be with 104To 109Individual cell, more specifically 104To 106Individual cell applies transformed cells.In general, between certain
Every applying, immunogene is multiple, until antibody titer increase.For example, in the case of short-term immunity process, can be with 2 to 4 days, spy
It is not that transformed cells are applied at the interval of 3 days, and antibody-producing cell can be collected after 2 to 3 administrations.Or with about every
After Zhou Yici interval is applied 5 to 6 times, antibody-producing cell is collected.
In the present invention, the antibody-producing cell of clone collection is to obtain monoclonal antibody.In order to be cloned, preferably
Immortalized antibody generation property cell.For example, cell fusion process such as hybridoma process or utilization Epstein-Barr virus (Epstein-Barr
Virus) conversion of (EBV) can be used as the process for immortalized antibody generation property cell.
In antibody-producing cell, a cell produces a type of antibody.Therefore, if can establish (for example, gram
It is grand) be derived from the cell mass of a cell, then it can obtain monoclonal antibody.Hybridoma process refers to antibody-producing cell wherein
The process for merging, immortalizing and cloning with appropriate cell strain.Immortalized antibody generation property cell can utilization technology（Such as have
Limit dilution method）To clone.Many cell strains available for hybridoma process are known.These cell strains have different
Genetic marker, the genetic marker are outstanding in terms of the immortalization efficiency of the cell based on lymphocyte and are selected at
Necessary to the cell to be succeeded in cell fusion.In addition, when being intended to obtain antibody-producing cell, it also can be used and lack
The cell strain of weary antibody producing ability.
For example, mouse myeloma P3x63Ag8.653 (ATCC CRL-1580) and P3x63Ag8U.1 (ATCC CRL-
1597) it is widely used as the cell strain for being used for cell fusion process in mouse and rat.In general, hybridoma passes through
Homologous cell is merged to prepare, but monoclonal antibody is available from closely related heterologous Hybrid knurl
(heterohybridoma)。
The concrete scheme of cell fusion is known.That is, by the antibody-producing cell of the animal through immunity inoculation with fitting
When fusion partner mix to carry out cell fusion.For antibody-producing cell, splenocyte, the leaching collected from lymph node are used
Bar cell, peripheral blood B cell etc..As fusion partner, the various cell lines having already mentioned above can be used.Melt to carry out cell
Close, use polyethylene glycol method or electro fusion method.
The cell to be succeeded subsequently, based on the selected marker selection being had by fused cell in cell fusion.For example,
In the case where being used for cell fusion when HAT sensitivity cell strains, pass through the cell for selecting to grow in HAT culture mediums
To select the cell to be succeeded in cell fusion.In addition, it is thus identified that antibody has desired as caused by the cell selected
Reactivity.
Reactivity based on antibody screens each hybridoma.That is, it is logical to produce the hybridoma of the antibody with reference to people PTPRS
Cross the above method and carry out selection.Preferably, the hybridoma of selection is subcloned, ought finally confirm situation caused by target antibody
Under, it is selected as the hybridoma for producing the monoclonal antibody of the present invention.
Specifically, can be selected based on the reactivity of the reactivity with people's cell or the transformed cells with expressing people's PTPRS genes
Select target hybridoma.It can be detected with reference to the antibody of cell by the principle of immunoassays.For example, it is used as antigen by the use of cell
ELISA can be used for detecting target antibody.Specifically, by the culture supernatant of hybridoma with being secured thereon as immunogene
The holder of people pDC or transformed cells contacts.In the case where including target antibody when culture supernatant, antibody is fixed on branch
Hold the cell capture on thing.Then, solid phase is separated with culture supernatant, washed if necessary, is caught so as to detectable
Obtain the antibody in solid phase.The antibody for identifying antibody can be used to detect antibody.For example, using anti-mouse immunoglobulin
Antibody test mouse antibodies.If the antibody of advance marker recognition antibody, its detection is then easy., can profit as label
With enzyme, fluorchrome, luminescent pigment etc..
On the other hand, as the holder for fixing cell, using particle, the inwall of microtiter plate.It can lead to
Cross on the surface of the container in particle or plastic manufacturing and carry out physical absorption to fix cell.For example, by polystyrene manufacture
Bead or reaction vessel can be used as fixing the holder of cell.
In the selection of hybridoma, it is contemplated that produce in some cases not for people PTPRS but for use as immunogene
The antibody of the host cell of transformed cells.For example, as in the embodiment shown, as immunogene and mouse is used as when by people's cell
When the animal of immunity inoculation, people's cell is identified as foreign substance, it is contemplated that generation combines its antibody.Present invention aims at
Obtain identification people PTPRS antibody.Therefore, it is not necessary to obtain the antibody of people's cell antigen of the identification in addition to people PTPRS.In order to pass through
Screening excludes the hybridoma of antibody as generation, can adsorb unexpected antibody in advance before the reactivity of antibody is confirmed.
It is the unexpected antibody of Antigen adsorption that desired antibody is combined that it can be utilized, which to exist,.Specifically, it is for example, anti-
The antibody of people's cell antigen in addition to people PTPRS can not wherein be detected the cell absorption of people PTPRS expression.In this hair
In bright, the host cell as immunogene is preferably used for adsorbing the antigen of unexpected antibody.
If necessary, active actual work of the monoclonal antibody (its binding activity to antigen has been identified) to pDC is confirmed
With.Effect to pDC can confirm for example, by following methods.
The culture that can be obtained from the hybridoma that monoclonal antibody is produced by cultivating collects the monoclonal antibody of the present invention.
Can in vitro or In vivo culture hybridoma.In vitro culture hybridoma can be come by using known culture medium such as RPMI1640.Training
Support in supernatant, accumulate the immunoglobulin secreted by hybridoma.Therefore, monoclonal antibody of the invention can be cultivated by collecting
Supernatant and purifying if necessary obtains.The purifying of immunoglobulin is more held in the case where serum ought not be added into culture medium
Easily.However, in order to quickly breed hybridoma and accelerate the generation of antibody, about 10% hyclone can be added to culture medium.
Can also In vivo culture hybridoma.Specifically, can be by hybridoma by the way that hybridoma is seeded in the abdominal cavity of nude mouse
Culture is in abdominal cavity.Monoclonal antibody accumulates in ascites.Therefore, it is desirable to monoclonal antibody can be by collecting ascites and necessity
Shi Chunhua is obtained.The monoclonal antibody obtained suitably can be modified or processed according to purpose.
The monoclonal antibody of the present invention can be inserted by the cDNA for the antigen binding domain that encoding antibody is obtained from hybridoma
Enter appropriate expression vector to express.Expressed for the cDNA of the variable region that obtains encoding antibody and in appropriate host cell
Technology be known.In addition, for the variable region including antigen binding domain to be bound into constant region to form chimeric antibody
Technology is also known.
For example, the preferred monoclonal antibody as the present invention, it is possible to provide the monoclonal antibody as caused by following hybridoma：With
Hybridoma 9H5-4, the hybridoma 10F7- with accession number FERM BP-11357 preservations of accession number FERM BP-11356 preservations
38th, with the hybridoma 13G5-52 of accession number FERM BP-11358 preservations, the hybridoma with accession number FERM BP-11359 preservations
13G5-57, with the hybridoma 14A8-85 of accession number FERM BP-11360 preservations, with accession number FERM BP-11361 preservations
Hybridoma 22H8-84, with the hybridoma 49F2-30 of accession number FERM BP-11362 preservations or with accession number FERM BP-11363
Hybridoma 55E7-79 of preservation etc..
The CDR for forming variable region humanized antibody, tool have been transplanted as the chimeric antibody including variable region or to it
The antibody for having the constant region from IgG or IgM is covered in the preferred antibody of the present invention.Present inventors have demonstrated that
The CDC that anti-PTPRS monoclonal antibody has anti-PTPRS expressivities cell is acted on.Therefore, have constant from IgG or IgM
The antibody in area has the cytotoxic effect by anti-PTPRS expressivities cell caused by CDC effects.This antibody-like can be used for pressing down
PTPRS expressivities cell processed such as pDC cell number.
The chimeric antibody or humanized antibody for identifying people PTPRS can be by genetic engineerings, by using the more of encoding antibody
Nucleotides produces.
Pass by about 4 since the structure that people PTPRS is illustrated in comfortable WO2007/041317 (JP2009-510102A)
Year；However, yet obtain can specific recognition people PTPRS antibody.Identify people PTPRS antibody first by the immune of the present invention
Original provides.That is, the invention provides identification people PTPRS antibody, it can be obtained by following process：
(1) protein for the extracellular domain for including people PTPRS is applied to the animal for treating immunity inoculation；
(2) antibody of the antibody with reference to people PTPRS is produced from the antibody-producing cell selection of the animal through immunity inoculation
Generation property cell；With
(3) antibody-producing cell of culture selection in (2), and the antibody for identifying people PTPRS is collected from culture.
It is specific expressed in people pDC people PTPRS has been illustrated.It is specific expressed also in gene expression analysis in people pDC
Confirmed in analysis by the present inventor using SAGE.However, in past report, in all situations based on mRNA points
People PTPRS expression is analysed.Make it possible to detect people PTPRS antibody by it due to not providing, the past does not analyze egg
The expression status of white matter.The antibody of combination people PTPRS provided by the present invention extracellular domain realizes people's PTPRS albumen
Analysis.
According to the actual confirmation made by the present inventor, the extracellular knot of the combination people PTPRS based on the present invention
Detect people pDC the monoclonal antibody specificity in structure domain.That is, the present invention relates to the side for detecting plasmacytoid dendritic cells
Method, it include making the monoclonal antibody of the extracellular domain with reference to people PTPRS or the fragment including its antigen binding domain with by
Cells contacting is tried, and detection has been bound to the monoclonal antibody of cell or the fragment including its antigen binding domain.
By detecting people PTPRS based on the present invention, it can confirm whether specific cells are pDC.That is, the invention provides use
People PTPRS identifies pDC method as index (index).Or can be by separating wherein according to the invention detects that people
PTPRS cell separates people pDC.That is, the invention provides methods of the user PTPRS as index separation pDC.
In the present invention, can mark in advance with reference to the monoclonal antibody of people PTPRS extracellular domain or anti-including it
The fragment of former land.For example, it can easily detect antibody by using luminescent pigment or fluorochrome mark.More specifically,
Make the antibody of fluorochrome mark with may be contacted including pDC cell aggregate, so as to use fluorchrome as index
To detect the cell that the antibody of the present invention is combined.If in addition, having separated the cell for wherein having detected that fluorchrome, can divide
From pDC.This series methods can easily be carried out by FACS principle.
Or can be in advance by the antibody binding of the present invention to solid support such as magnetic-particle.It is bound to solid phase support
The antibody identification people PTPRS of thing, and pDC is captured by solid support.Therefore, it can detect and separate pDC.
Antibody needed for detection pDC based on the present invention can be provided as the reagent for detecting pDC.That is, it is of the invention
The reagent for detecting pDC is provided, it includes the monoclonal antibody of the extracellular domain with reference to people PTPRS or including its antigen
The fragment of land.For the reagent for being used to detect pDC of the present invention, in addition to antibody, positive control or feminine gender can be combined
Control.Can for example, expressing the transformed cells (it is used as immunogene) of people PTPRS extracellular domain, the pDC collected from people etc.
As positive control.Normally, few people pDC can be only obtained from peripheral blood.Therefore, transformed cells are particularly preferably used as this hair
The positive control of bright reagent.On the other hand, any cell for not expressing people PTPRS can be used as negative control.
That is, the present invention is provided to detect people pDC kit, it includes the extracellular domain with reference to people PTPRS
Monoclonal antibody or the fragment including its antigen binding domain.
In addition, the present inventor has analyzed work of the antibody to pDC of the people PTPRS of combination extracellular domain
With.Therefore, they have confirmed that the antibody with reference to people PTPRS extracellular domain suppresses pDC activity.That is, the present invention relates to
For suppress interferon produce property cell active method, it include will be following in any component contacted with pDC：
(a) people PTPRS is combined to suppress pDC active monoclonal antibody or fragment including its antigen binding domain, and
(b) immunoglobulin of the complementary determining region of the monoclonal antibody of (a) has been transplanted to it or including its antigen knot
Close the fragment in area.
Or the present invention relates to the active method for suppressing pDC in live body, methods described is included under being applied to live body
Any component in row component：
(a) people PTPRS is combined to suppress pDC active monoclonal antibody or fragment including its antigen binding domain,
(b) immunoglobulin of the complementary determining region of the monoclonal antibody of (a) has been transplanted to it, or including its antigen knot
The fragment in area is closed, and
(c) polynucleotides of the component described in (a) or (b) are encoded.
In the present invention, pDC refers to the ability for producing IFN, and in cell surface upper table intelligent PTPRS cell.
Hereinafter, unless otherwise stated, otherwise pDC not only includes the cell of the precursor as dendritic cells but also including tool
There is generation IFN ability and in cell surface upper table intelligent PTPRS cell.For identifying that such pDC method is known
's.For example, several cell surface markers can be used to distinguish pDC and other blood cells as mark.Specifically, people pDC
Cell surface marker characteristic spectrum following (Shortman, K. and Liu, YJ, Nature Reviews2：151-161,2002).
BDCA-2 positive cells are there has been reported in recent years is positioned as pDC (Dzionek, A. et al. J.Immunol.165：6037-
6046,2000)。
[characteristic spectrum of people pDC cell surface antigen]
CD4 is positive, CD123 positives pedigree (CD3, CD14, CD16, CD19, CD20, CD56) is negative, CD11c is negative
Therefore, the expression characteristic spectrum with these known marks and the cell also with the ability for producing IFN also can quilts
Referred to as pDC.In addition, the cell of the characteristic spectrum of the even one group expression pattern with the expression characteristic spectrum for being different from these marks,
There is the cell for the ability for producing IFN, the cell is covered in pDC in live body.
In addition, as the feature observed generally in people pDC, following features can be shown.
[features of cellular forms]
- it is similar to thick liquid cell.
- it is the round cell with smooth cell surface.
- it has relatively large nucleus.[functional character of cell]
- it produces large amounts of type i IFN in a short time in virus infection.
- its virus infect after be divided into dendritic cells.
In the present invention, pDC active suppression refers to suppress at least one function being had by pDC.Work(as pDC
Energy, IFN generation and cell survival can be shown.In other words, cell survival can be considered as cell number.
Therefore, the suppression of one or two of these functions refers to pDC active suppression.The I as caused by pDC is illustrated
Type IFN gives rise to diseases.Therefore, its can as those diseases therapeutic strategy be used for suppress pDC cell number and
IFN generation.
For example, indicate the relation between the pathological condition of autoimmune disease and IFN α.Most of IFN αs are produced by pDC
It is raw.Therefore, it is suppressed if it is produced, the pathological state brought by IFN α can be mitigated.Meanwhile in the present invention, pDC pairs
Suppress to refer to suppress the generation that the IFN as caused by pDC at least one of works as the IFN of type caused by IFN.I types IFN is this hair
Bright preferred IFN.Among these IFN, IFN α is important.
That is, the present invention relates to for suppressing reagent caused by IFN, it includes the extracellular domain with reference to people PTPRS
Antibody is as active component.Or the invention provides the caused method for suppressing IFN, methods described, which includes applying, to be tied
Close the antibody of people PTPRS extracellular domain.In addition, the present invention relates to the antibody for the extracellular domain for combining people PTPRS
For producing the purposes for the caused pharmaceutical composition for being used to suppress IFN.
PDC includes the cell for producing a large amount of IFN as caused by a few cell.For example, the dendron with stimulations such as viruses is thin
The precursor of born of the same parents produces most of IFN as caused by live body.Produce the suppression of a large amount of IFN pDC cell number thus lead
Cause the suppression of IFN yield.Therefore, the pathological condition brought by IFN α can also be mitigated by suppressing pDC cell number.
In a preferred embodiment of the invention, it was confirmed that Anti-Human's PTPRS monoclonal antibody combination people's PTPRS expressivities
Cell and pass through CDC (complement dependent cytotoxicity) effect assign cytotoxic effect.CDC effects are the important of antibody agent
One of mechanism of action.The Anti-Human PTPRS monoclonal antibodies of the present invention also have by anti-human PTPRS caused by its CDC effects
Expressivity cell（Such as pDC）Strong cytotoxicity effect.That is, except suppression mechanism caused by IFN in preferred embodiments with
Outside, it is contemplated that by suppressing effect caused by IFN caused by anti-pDC cytotoxic effect.
The anti-of identification people PTPRS used in present invention extracellular domain can be obtained based on previously described method
Body.The antibody of the present invention can be any classification.In addition, the biological species that antibody is derived from is also unrestricted.In addition, bag
The fragment for including the antigen binding domain of antibody can be used as antibody.For example, by including antigen-binding portion caused by IgG enzymatic digestion
The antibody fragment of position may also used as the antibody of the present invention.Specifically, can disappearing by using papain or pepsin
Change to obtain antibody fragment such as Fab or F (ab') 2.It is well known that this kind of antibody fragment can be used as with the knot for antigen
Close the antibody molecule of affinity.Or also can be used by genetic recombination the antibody that builds, as long as its maintain necessary to antigen
Binding activity.By genetic recombination build antibody example may include chimeric antibody, CDR grafted antibody, scFv, binary and
Linear antibodies and the multi-specificity antibody that is formed by antibody fragment etc..For obtaining the side of this antibody-like based on monoclonal antibody
Method is known.
In the present invention, if necessary can modified antibodies.According to the present invention, identification people PTPRS extracellular domain resists
Body has the function that to suppress the active of pDC.I.e., it is contemplated that antibody has the possibility of anti-pDC cytotoxic effect in itself.It is aobvious
The Subclass of antibody for showing strong effector function is known.Or modified by using cytotoxic substance (cytotoxic agent) anti-
Body, the active effect for suppressing pDC can be further enhanced.
The example of cytotoxic substance may include following material.
Toxin：Pseudomonas endotoxin (PE), diphtheria toxin
Lysine
Radioisotope element：Tc99m、Sr89、I131、Y90
Anticancer：Calicheamicin, mitomycin C, taxol
The toxin being made up of protein is bound to antibody or its fragment etc. using bifunctional reagent.Or it will can compile
The gene of code toxin is connected to the gene of encoding antibody to produce the fusion protein of two genes.For radio isotope is first
The method that element is bound to antibody is also known.For example, by using chelating agent, radioisotope element labelled antibody is utilized
Method be known.In addition, anticancer can be bound to by antibody by using sugar chain or bifunctional reagent.
In the present invention, its structure also is used as active component by manually modified antibody.For example, for improving antibody
Cytotoxic effect and the various method of modifying of stability be known.Specifically, the sugar chain of wherein heavy chain has been modified
Immunoglobulin is known (Shinkawa, T. et al., J.Biol.Chem278:3466-3473.2003.).By modifying sugar
Chain, ADCC (Antibody -dependent cell cytotoxicity) activity of immunoglobulin are strengthened.
When the antibody for combining people PTPRS extracellular domain is contacted with pDC, its activity inhibited.Therefore, it is this kind of
Antibody can be used for being used for the active reagent or method for suppressing pDC.That is, the invention provides the active examination for suppressing pDC
Agent, the reagent include the component selected from following (a)-(c) of at least one type as active component.Or the present invention relates to
And for suppressing pDC active method, methods described includes applying at least a type of group selected from following (a)-(c)
Point.In addition, it is used for the purposes for producing the active reagent for being used for suppressing pDC the present invention relates to the component selected from following (a)-(c).
(a) antibody of people PTPRS extracellular domain or the fragment including its antigen binding domain are combined, and
(b) immunoglobulin of the complementary determining region of the monoclonal antibody of (a) has been transplanted to it, or including its antigen knot
Close the fragment in area.
In the present invention, as the active monoclonal antibody for suppressing pDC, using identification people PTPRS extracellular knot
The monoclonal antibody in structure domain.In the present invention, using a type or the monoclonal antibody of multiple types.For example, can will be more
The monoclonal antibody of the identification people PTPRS of individual type extracellular domain is incorporated to and in the present invention.
Antibody, which can be confirmed as follows, has the function that the IFN generation property activity for suppressing pDC.PDC is produced by the stimulation of virus
A large amount of IFN.Anti- pDC antibody is provided by before or after being stimulated using virus, or while with being stimulated using virus, and
Using the pDC that antibody is not provided it as control, compare the ability for producing IFN.It can be included in by measurement in pDC culture
IFN-α and IFN-β in clear liquid produce IFN ability to assess.As result of the comparison, when the IFN in supernatant amount because
Addition antibody and when substantially reducing, can the antibody of exact p-value there is the ability for suppressing generation IFN.It is such for measuring
IFN method is known.PDC is the cell that most of IFN is produced in live body.Therefore, produce IFN's by suppressing pDC
Ability, it can adjust the caused state of IFN in live body.
In the present invention, pDC activity includes the maintenance of pDC cell number.Therefore, pDC of the invention activity suppression
System includes suppressing pDC cell number.If it is confirmed that pDC cell number is suppressed in the presence of antibody, then find
Antibody suppresses pDC activity.As the control for comparing, the inertia from animal can be used as in IFN generation
Immunoglobulin, the animal that the inert immune globulin is derived from and the animal for its activity antibody to be confirmed
Species are identical.The cell number for the number quantitative comparison pDC for calculating cell can be passed through.It can be calculated using FACS or microscope thin
Born of the same parents' number.
In addition, as by the result of the infection of virus etc., it is additionally considered that pDC is divided into the induction of referred to as DC2 (dendritic cells 2)
Th2 cell.If it can suppress to produce by the IFN of pDC caused by virus stimulation, it is also possible to which can suppress differentiation turns into Th2.
Therefore, it is expectable to various allergic disease (allergy for monoclonal antibody of the invention caused by suppression IFN
Disease therapeutic effect).
When the extracellular structure that identification people PTPRS is applied to host's (it is different from the organism species that antibody is derived from)
In the case of the antibody in domain, it is expected for antibody to be processed into the shape for being hardly identified as foreign substance by host.For example, by adding
Into following molecule, immunoglobulin variable must be difficult to be identified as foreign substance work.For processing immunoglobulin molecules as follows
Technology be known.
Fragment (the Monoclonal Antibodies comprising antigen binding domain of-shortage constant region：Principles
And Practice, the 3rd edition, Academic Press Limited.1995;Antibody Engineering,A
Practical Approach,IRL PRESS,1996)
- by monoclonal antibody antigen binding domain and host immunoglobulin the chimeric antibody that forms of constant region
(Experimental Manual for Gene Expression, Kodansha Ltd., 1994 (by Isao Ishida and
Tamie Ando write))
- the complementary determining region (CDR) replaced by using the CDR of monoclonal antibody in the immunoglobulin of host obtains
(Experimental Manual for Gene Expression, Kodansha Ltd., 1994 (by Isao for CDR displacements antibody
Ishida and Tamie Ando write)).
Or the immune globulin variable region gene of people can obtain (McCafferty J. etc. by phage display
People, Nature348：552-554,1990；Kretzschmar T et al., Curr Opin Biotechnol2002Dec：13
(6)：598-602.).In phage display, the gene integration of encoding human immunoglobulin variable region is entered into bacteriophage base
Cause.Phage library can be prepared by using various immunoglobulin genes as source.Variable region is expressed as by bacteriophage
Form the fusion protein of the protein of bacteriophage in itself.By the variable region maintenance on the phage surface of phage expression and antigen
Binding activity.Therefore, the bacteriophage of cell or the antigen for having expressed antigen etc. is combined by selection, can be sieved from phage library
The bacteriophage of the variable region with desired binding activity has been expressed in choosing.
In addition, the gene for encoding the variable region with desired binding activity is retained in what is selected by such mode
In phage particle.That is, in phage display, the gene of variable region of the coding with desired binding activity can be by making
Obtained by the use of the binding activity of variable region as index.
According to the present invention be used for suppress pDC active reagent or method, can will identify that people PTPRS's is extracellular
The antibody of domain or antibody fragment including at least its antigen binding domain are as protein or the multinuclear of code for said proteins
Thuja acid is applied.In order to using polynucleotides, it is expected to utilize following carriers：The nucleotides for encoding desired protein is placed in suitable
When promoter control under, in order to express desired protein.Enhancer or terminator can be also placed on carrier.Can
Retain form the heavy chain of immunoglobulin and the gene of light chain and can the carrier of expressing immunoglobulin molecule be known.Can
The carrier of expression immunoglobulin can be applied by introducing cell.In the administration to live body, can as former state apply can by
Live body is applied to pass to the carrier of cell.
Or carrier can be introduced to the lymphocyte once separated with live body body, then return it into live body (in vitro).
According to the present invention be used for suppress pDC active reagent or method, have to be used as immunoglobulin to living
The amount for the monoclonal antibody that body is applied is usually per kilogram of body weight 0.5mg to 100mg, such as per kilogram of body weight 1mg to 50mg, excellent
Select per kilogram of body weight 2mg to 10mg.The interval to live body administration of antibodies can be suitably adjusted, in order to living during the maintaining treatment phase
The valid density of immunoglobulin in body.Specifically, can be with the interval administration of antibodies in 1 to 2 week.Route of administration is optional
's.Those skilled in the art can properly select effective route of administration for therapy.Specifically, can show oral or parenteral
Using.For example, can be systemic by intravenous injection, intramuscular injection, intraperitoneal injection or hypodermic injection etc. or local application resists
Body.The example of preparation in the present invention suitable for parenteral administration may include injection, suppository, aerosol etc..Will be anti-in addition, working as
When body is supplied to cell, there is provided usually 1 μ g/mL, preferably 10 μ g/mL or more, more preferably 50 μ g mL or more, more preferably
0.5mg/mL or more immunoglobulin.
In being used to suppress pDC active reagent or method according to the present invention, it can be applied by any method to live body
Monoclonal antibody.In general, monoclonal antibody is mixed with pharmaceutically acceptable carrier.If necessary, can be to the Dan Ke
Blast blending such as thickener, stabilizer, preservative and solubilizer in grand antibody.The example of such carrier or additive can
Including lactose, citric acid, stearic acid, magnesium stearate, sucrose, starch, talcum, gelatin, agar, vegetable oil, ethylene glycol etc..Term
" pharmaceutically acceptable " refers to by the regulator of each national government be received, or is listed in each national pharmacopeia
In or in animal, mammal, particularly using in the pharmacopeia being commonly recognized in people.For suppressing the present invention's
PDC active reagent can exist in the form of lyophilized pulvis or tablet (including a dosage or multiple dosage).It can also incite somebody to action
Lyophilized pulvis or tablet and injectable sterilized water, physiological saline or buffers combinations are for dissolved composition, so as to apply
With providing desired concentration before.
In addition, when when to be applied in the form of the carrier for expressing immunoglobulin, it is contemplated that can be using heavy chain and light chain as dividing
The plasmid co-transfection opened, can be with per kilogram of body weight 0.1 to 10mg, such as per kilogram of body weight 1 applies each plasmid to 5mg.
In addition, in order to introduce cell in vitro, 1 to 5 μ g carrier/10 are used6Individual cell.
Hereinafter, the present invention is explained in greater detail by reference to embodiment.
All prior art literatures cited herein are incorporated herein by reference.
Hereinafter, by reference to embodiment, the present invention will be explained in further detail, but the present invention is not construed as being implemented
Example is limited.
Embodiment
Embodiment 1
The analysis of A.PTPRS expression
A-1 the analysis in SAGE libraries) is used
Utilize SAGETM(Serial Analysis of Gene Expression serial analysis of gene expression) method compare and
Gene is analyzed in person monocytic cell, pDC and utilizes the expression in the pDC of herpes simplex virus (HSV) processing.Analysis method is such as
Under.
Monocyte is separated into CD14 positive cells using cell sorter, and using pDC as BDCA-4 positive cells
Separated with human peripheral blood mononuclear cell.In addition, pDC12 hours are cultivated in the presence of HSV to prepare the pDC of activation.From
Each cell obtains RNA, by using I-SAGETMKit (Invitrogen) prepares SAGE libraries.Utilize SAGE analysis softwares
(Invitrogen) the base sequence data that are obtained of the analysis with about 100000 labels.As a result, it is as having
The gene of 0/7/0 monocyte/pDC/pDC+HSV score value（Show the specific expressed genes of pDC）, it was found that
The gene known：PTPRS(GenBank Acc#NM_002856.3).PTPRS is by SEQ ID NO:Base sequence shown in 2 is compiled
Code.In addition, it is that have immunoglobulin like domain (Ig- spline structures domain) and fi-bronectin type III in extracellular space
The single span spanning domain in spline structure domain.In addition, it has two Protein-tyrosine-phosphatase regions (PTP is tied in region in the cell
Structure domain) (Fig. 1).
A-2) the expression by quantitative RT PCR analysis PTPRS mRNA in different people's immunocompetent cells
Expression of the PTPRS in immunocyte is analyzed in more detail.It is each from human peripheral separation by cell sorter
Individual cell.RNA is extracted from each cell colony of separation, synthesizes cDNA.Using the cDNA of acquisition as template, according to general
Method carries out quantitative RT-PCR to analyze PTPRS mRNA expression.GAPDH (the 3- phosphorus by using known constant expressed
Acid glycerol aldehyde dehydrogenase) expression of gene is normalized, the expression of the PTPRS genes between relative immunity cell.
The base sequence of used primer and as follows for PCR condition.
PTPRS forward primer：5'CAC GGC CTATGA CCT CCA3'(SEQ ID NO:3)
PTPRS reverse primer：5'AAG TTC TTG GGC GAG ACT TG 3'(SEQ ID NO:4)
GAPDH forward primer：5'CCA CCC ATG GCAAAT TCC3'(SEQ ID NO:5)
GAPDH reverse primer：5'TGG GAT TTC CAT TGATGA CAA G 3'(SEQ ID NO:6)
1 circulation, is carried out 2 minutes at 50 DEG C,
1 circulation, is carried out 10 minutes at 95 DEG C, and
50 circulations [carry out 15 seconds at 95 DEG C, and carried out 60 seconds at 60 DEG C].
Analyze monocyte, pDC, using HSV stimulate pDC, B cell (CD19+ cells), T cell (CD3+ cells),
Using the T cell and NK cells (CD56+ cells) of PMA (phorbol 12-myristinate 13- acetic acid esters) activation stimulated, show
Show that PTPRS is expressed with pDC specificity patterns.It moreover has been found that following feature：The pDC that PTPRS expression is utilized HSV stimulations subtracts
Few (Fig. 2).
A-3) the expression by quantitative RT PCR analysis PTPRS mRNA in people organizes
In addition, using ABI PRISM7000 (Applied Biosystem), pass through the table in quantitative PCR research tissue
Reach.As cDNA groups (panel), BD is usedTMMore tissue cDNA group (the people I of MTC；Catalog number (Cat.No.) 636742, people is immunized；Catalogue
Numbers 636748, human blood fraction；Catalog number (Cat.No.) 636750；It all is from Becton Dickinson).The base of used primer
Sequence is shown in following.
PTPRS forward primer：5'ACT CAC CCA CAC CCT ACA AGA 3'(SEQ ID NO:7)
PTPRS reverse primer：5'CTT GGT GGT ACG GCC ATC3'(SEQ ID NO:8)
GAPDH forward primer：5'CCA CCC ATG GCAAAT TCC3'(SEQ ID NO:5)
GAPDH reverse primer：5'TGG GAT TTC CAT TGA TGA CAA G 3'(SEQ ID NO:6)
By using SYBR green PCR master mix kits (Applied Biosystem), using can slave phase
The ABI PRISM7000 obtained with company enter performing PCR.It can be used for from the sequence detection system software that same companies obtain point
Analysis.Reaction condition is as follows.
Step 1：1 circulation, is carried out 2 minutes at 50 DEG C
Step 2：1 circulation, is carried out 10 minutes at 95 DEG C
Step 3：40 circulations, carry out 15 seconds at 95 DEG C and carried out 1 minute at 60 DEG C
Normalizing is carried out by using the expression of GAPDH (glyceraldehyde 3-phosphate dehydro-genase) gene of known constant expression
Change, compare the expression of the PTPRS genes between tissue.As a result, PTPRS mRNA widely express (Fig. 3) in the tissue.
The preparation of B.PTPRS expression vectors
In order to express PTPRS albumen, the preparation of the expression vector of PTPRS genes is carried out.It is only that PTPRS genes is whole from
Close into pCR4-TOPO cloning vectors (Open Biosystem cc#MHS1010-98052887) and be integrated into
Taken out in the PTPRS cDNA clones of pcDNA3.1 expression vectors (PTPRS/pcDNA3.1).By using the PTPRS/ of acquisition
PcDNA3.1 plasmids are as template, using the primer comprising EcoRI, Not I and Kozak sequences (GCC GCC ACC) (on drawing
The information of thing is shown in hereinafter) amplification PTPRS genes.PCR primer is cloned into pMX-IP in EcoRI and Not I sites to reverse
Record in viral vector (PTPRS/pMX-IP).In order to enter performing PCR reaction, the KOD Plus archaeal dna polymerases of a unit are used
(TOYOBO), reaction condition is that 1 circulation (being carried out 2 minutes at 94 DEG C) and 25 circulations [are carried out 15 seconds and at 68 DEG C at 94 DEG C
Carry out 30 seconds 4 minutes].
Forward primer (SEQ ID NO:9)：5'aaa GAA TTC gcc gcc acc ATG GCG CCC ACC TGG
GGC CCT3'
Reverse primer (SEQ ID NO：10)：5'aaa gcg gcc gcT TAG GTT GCA TAG TGG TCAAAG
C3'
In above-mentioned base sequence, lowercase character represents Restriction Enzyme EcoRI cleavage site or Not I site.5'
The aaa of end is the Extra bases for enzymatic cutting.
C. the preparation of people PTPRS (hPTPRS) expressivity cell
In order to produce the retrovirus for including PTPRS genes, using FuGENE kits (Roche), PTPRS/ is utilized
PMX-IP and retrovirus package carrier PCL-ECO transiently transfects thin as the HEK-293T of the nephrocyte strain of Human embryo
Born of the same parents.Two days later, collecting wherein virus includes the cell culture supernatant of hPTPRS genes, is utilized as the spleen from BALB/c mouse
Dendritic cells D2SC/1 cells (this to be based on Paglia et al., J.Exp.Med., 178,1893-1901 (1993) to prepare)
Infected.Because pMX-IP Retroviral Vectors include puromycin resistance gene, by using puromycin culture infection
The cell that D2SC/1 cells only to express hPTPRS may survive, so that selection becomes possibility.Sorted and selected by FACS
HPTPRS expressivity D2SC/1 cells are selected, and are cultivated.In order to confirm hPTPRS expression, by 10 μ g/mL goat IgG
And the hPTPRS polyclonal antibodies (pAb that is obtained commercially (SantaCruz)；R＆D selected hPTPRS/D2SC/1 cells) are added to
(each 100 μ L), mixture is incubated 30 minutes in 4 DEG C.Cell is washed using PBS, then adds 50 μ L 100 times of dilution
The anti-anti-goat IgG antibody (SantaCruz) of FITC- marks, mixture is incubated 30 minutes at 4 DEG C.After being washed with PBS, profit
With FACSCalibur (BD) input data (Fig. 4).
Embodiment 2
A. the preparation of Anti-Human PTPRS monoclonal antibodies
A-1) it is immunized
As the cell as immunogene, above-mentioned hPTPRS/D2SC/1 cells are used.BALB/c mouse is anaesthetized, with every
Freund's complete adjuvant (CFA) emulsion is subcutaneously injected to foot pad 50 μ L of foot.Total amount is 100 μ L/ mouse.At second day, by making
The hPTPRS/D2SC/1 cells and incomplete Freund's adjuvant (IFA) for being used as immunogene preparation prepare emulsion, and it subcutaneous is noted
It is incident upon foot pad (50 μ L/ foots, the μ L/ mouse of total amount 100).Carry out immunity inoculation once within every two days, carry out 3 times altogether, at last
Collect draining lymph node (drawing lymph node) within 3 days after secondary immunity inoculation.
A-2) cell fusion
Draining lymph node is collected from two foots of the mouse through immunity inoculation, by it with including 10%FBS's
The murine myeloma cell P3-X63-Ag8.563 mixing of culture in RPMI1640 culture mediums (SIGMA) so that lymph node cells
Turn into 5 with the ratio of myeloma cell:4, by the way that cell is collected by centrifugation.PEG1500 (Roche) is used added to the cell of mixing
In cell fusion.The cell (hybridoma) of fusion is washed, the fused cell is incubated at comprising cell growth supplement+HAT
(Sigma)-RPMI1640 culture mediums (comprising 2mM Glus, 100 units/ml penicillin, 100 μ g/ml streptomysins,
10mM HEPES, 1mM Sodium Pyruvates, 50 μM of 2-ME) 10% hyclone (FBS) in.
A-3) the FACS screenings of the hybridoma carried out using the hPTPRS/D2SC/l cells through immunity inoculation
The anti-CD16/32 (2.4G2) that 50 μ L are prepared into 2.5 μ g/ml is added to 3x105The D2SC/1 cells in/hole or
HPTPRS/D2SC/1 cells are to close FC acceptors.After being washed with PBS, addition be prepared into 10 μ g/ml goat IgG, it is commercially available can
Anti- hPTPRS pAb (R＆D), mouse IgG2ak(BioLegend) and culture hybridoma culture supernatant (each 60 μ
L), mixture is incubated 60 minutes at 4 DEG C.After being washed with PBS, the FITC- for the diluting 50 times anti-goat IgGs marked are resisted
The anti-mouse IgG antibody (BD) of body and the PE- of dilution 100 marks is added to cell (each 50 μ L), by mixture in 4 DEG C of lucifuges
Incubate 30 minutes.After being washed with PBS, cell is suspended in 200 μ L PBS.Number is collected using FACS Calibur (BD)
According to.Collected data are developed to gate living cells by FSC and SSC two-dimentional point diagram (dot plot).Collect data until
The data of cell in the gate reach 2000 countings.As a result, 13 hybridization for producing anti-hPTPRS antibody can be obtained
Knurl (2G6,28G10,4B2,2G2,9H5,10F7,22H8,49F2,9D2,14A8,55E7,13G5,16H2) (Fig. 5).
A-4) screened using the FACS of CAL-1 cells
By 3x105The culture supernatant of above-mentioned each hybridoma of the personal pDC like cells strain CAL-1 cells in 50 μ L
In dyed, 4 DEG C carry out 15 minutes.Using FACS buffer solution (1%FBS+PBS) washing cell once, then centrifugation with except
Remove supernatant.Then the anti-mouse IgG antibody that 2 μ g/ml PE- is marked is reacted 20 minutes at 4 DEG C.Washed with FACS buffer solution
Wash cell once and centrifuge.Using FACS buffer solution resuspension cell precipitation, analyzed using Calibur.It is as a result, miscellaneous
Hand over 2G6,4B2,2G2,9H5,10F7,22H8,49F2,14A8,55E7,13G5 and 16H2 and CAL-1 in knurl culture supernatant
It is fully good.On the other hand, 28G10 and 9D2 reacts minimum (Fig. 6).
A-5) the FACS screenings carried out using human peripheral pDC
[separation of human PBMC]
20ml peripheral blood is collected from Healthy People, using HISTOPAQUE-1077 (SIGMA), is separated by centrifugation by specific gravity
PMBC (PBMC).Using each sample to 1x 106Individual PBMC is dyed.Washed using FACS buffer solution
Cell, with 25 μ L, 5 times of dilution addition Fc closed reagents (Miltenyi), reaction 15 minutes is carried out at 4 DEG C.Buffered with FACS
After liquid washing, 50 μ the L cell culture supernatant of every kind of hybridoma, 10 μ g/ml goat IgG, anti-hPTPRS pAb are added
With mouse IgG 2a, κ, reaction 20 minutes is carried out at 4 DEG C.After being washed with FACS buffer solution, 8 μ g/ml of addition FITC- marks
Anti- anti-goat IgG antibody or 2 μ g/ml PE- mark anti-mouse IgG antibody, carried out at 4 DEG C reaction 20 minutes.With
After FACS buffer solution washing, the anti-BDCA2 antibody that the APC- of 50 μ L 10 times of dilution is marked is reacted 20 minutes at 4 DEG C.With
After FACS buffer solution washing, cell is resuspended in 300 μ L FACS buffer solution, analyzed using FACS calibur.Make
For result, 2G6,28G10,4B2,2G2,9H5,10F7,22H8,49F2,14A8,55E7 and 13G5, which are shown, is specific to pDC cells
The association reaction of colony.9D2 shows the combination to pDC, and also display and the reaction of the cell mass in addition to pDC (BDCA2-).
16H2 does not show the reaction (Fig. 7) for PBMC.
Specific test to anti-PTPRS antibody
PTPRS belongs to PTPR families, and the amino acid sequence from its several family molecules has the amino for PTPRS
The high homology (Fig. 8) of acid sequence.
A-6) check whether the Hybridoma Cell Culture supernatant of 10 types only specifically binds PTPRS, the hybridization
Knurl culture supernatant produce identification PTPRS and specifically bind people pDC antibody (2G6,4B2,2G2,9H5,10F7,22H8,
49F2、14A8、55E7、13G5).By the way that the N-terminal of FLAG tag expressions to molecule is prepared with PTPRS with especially high same
PTPRA (40%), the PTPRD (76%) of source property and PTPRF (67%) cell through transfection, and the cell is dyed.Profit
Confirm expression of the hPTPRE in the cell of transfection with western blot, but not can confirm that the expression on cell surface.Cause
This, hPTPRE is not expressed on cell surface.As a result, 4B2 and hPTPRD reactions (Fig. 9 C), 2G6 is shown with hPTPRF's
Cross reactivity (Fig. 9 D).The antibody of other 8 types shows PTPRS specific bindings (Fig. 9 A-D).
A-7) cross reactivity of the anti-PTPRS antibody to monkey
Using HISTOPAQUE-1077 (SIGMA) by centrifugation by specific gravity from peripheral blood (10ml；Shin-Nippon
Biomedical Laboratories, Ltd.) separation machin PBMC.In order to carry out FACS, 5x10 is used per sample5It is individual
Cell.Cell is washed using FACS buffer solution, the 10 μ L 10% machin serum using FACS buffer solution dilution is added
Carried out 20 minutes at 4 DEG C to reaction wherein, is made.After being washed with FACS buffer solution, add 100 μ L each hybridoma it is thin
The mouse IgG 2a, κ or mouse IgG 1 of born of the same parents' culture supernatant and 10 μ g/ml, κ (BioLegend), reaction is set to carry out 15 points at 4 DEG C
Clock.After using FACS buffer solution washing, the anti-mouse IgG antibody (BD) of 1 μ g/ml of addition APC marks, make reaction at 4 DEG C
Carry out 20 minutes.After being washed with FACS buffer solution, diluted with 25 μ L, with 10 times, the anti-pedigree antibody for marking FITC-
(BD), the anti-CD123 antibody (BD) of PE- marks and the anti-HLA-DR antibody (BD) of PerCP7Cy5.5- marks are in 4 DEG C of reactions
15 minutes.After using FACS buffer solution washing, by Cell resuspension in 300 μ L FACS buffer solution, FACS is utilized
Calibur is analyzed.As the Hybridoma culture supernatants used, 7 kinds are have selected as PTPRS specificity and is tied well
Close CAL-1 cells and people pDC type：49F2,55E7,14A8,13G5,10F7,22H8 and 9H5.As a result, all hybridization
Oncocyte culture supernatant is specifically bound to the pDC population groups (pedigree-CD123+HLA-DR+) (Figure 10) of machin.
A-8) hybridoma is individualized
Collect each in the hybridoma (49F2,55E7,14A8,13G5,10F7,22H8 and 9H5) of above-mentioned 7 types
Kind, it is suspended in sorting buffer solution (1%FBS/PBS) with as 1x105Individual cell/ml.By using FACS Aria
(BD) unicellular sorting, is carried out.Data are collected, pass through X-axis：FSC and Y-axis：The data that SSC two-dimentional point diagram exploitation is collected.
Living cells is gated in two-dimensional points.The gate for removing doublet from the cell in living cells door is carried out, cell colony is allocated
To 96 hole flat undersides with as 1 cells/well.The cell culture of unicellular sorting will be experienced in HAT culture mediums (RPMI1640
+ 2mM Glus, 100 units/ml ampicillins, 100 μ g/ml streptomysins, 10mM HEPES, 1mM Sodium Pyruvates and
50 μM of 2-ME) in+hybridomas grew replenishers HFCS (Roche).Hereafter, entered by using the cell culture supernatant of hybridoma
The dyeing of row D2SC cells and hPTPRS/D2SC cells (Figure 11 A and B), CAL-1 cells (Figure 11 C) and people pDC (Figure 11 D), choosing
Select single hybridoma.
Embodiment 3
The purifying of antibody
Obtained using Protein G Sepharose FastFlow (GE Healthcare) by purifying from the culture supernatant of hybridoma
8 types antibody purification (9H5-4,10F7-38,13G5-52,13G5-57,14A8-85,22H8-84,49F2-30 and
55E7-79).By using Pierce rapid ELISA mouse mAb Isotyping kits (Thermo Fisher
Scientific), isotype is determined.As a result, 13G5-52 and 13G5-57 are mouse IgG 2b, κ, 55E7-79 have mouse
IgG2b, κ and mouse IgG 1, both κ, other is mouse IgG l, κ.If the antibody of purifying includes endotoxin, it can influence
The result of property measure test.Therefore, endotoxic concentration is measured.Used kit is Endospecy ES-50M
Set, Toxicolor DIA-MP set and Endotoxin standard product CSE-L set (all by
Seikagaku Biobusiness Corporation are produced).As a result, all purifying antibody all have be equal to or
Endotoxin concns (Figure 12) less than standard value 0.3EU/mg Ab.
Reactive research on the antibody of purifying
The binding ability (Figure 13) of the antibody of purifying is confirmed using people's pDC- like cells strain CAL-1 cells.In addition, institute
There is the binding ability (Figure 14) that antibody maintains the people pDC colonies (BDCA2+) for human peripheral.
People PTPRS is about 96% for the homology of mouse PTPRS (mPTPRS) amino acid sequence.Due to them each other
It is significantly similar, whether the Anti-Human PTPRS antibody of preparation is have studied also in relation with mouse PTPRS.Utilize each anti-of 10 μ g/ml
PTPRS antibody is to the wherein Chinese hamster ovary celI (Chinese hamster ovary cell of forced expression mPTPRS gene；Hereinafter referred to as
MPTPRS/CHO) dyed.Cell number is 2x 105/ sample.After using FACS buffer solution washing, by PE- marks
Anti-mouse IgG antibody dilutes 50 times, is dyed with 25 μ L.As a result, 49F2-30,13G5-52,13G5-57 and 22H8-
84 are bound to mPTPRS/CHO (Figure 15).
Embodiment 4
Complement dependent cytotoxicity of the anti-PTPRS antibody to hPTPRS expressivity cells
By using young rabbit complement, anti-expression people PTPRS Chinese hamster ovary celI is measured (hereinafter referred to as hPTPRS/
CHO) and the anti-PTPRS antibody of mouse PTPRS/CHO cells (hereinafter referred to as mPTPRS/CHO) complement dependent cellular
Toxicity (hereinafter referred to as CDC activity).Calculated by using the measured value of the lactase dehydrogenase (LDH) by being discharged from cell
Cytotoxicity obtain activity as index.By each cell with 2x 104The μ L/ holes of individual cell/50 are distributed to 96 hole U-shapeds
Bottom plate.Utilize CDC culture mediums (+100 units of RPMI1640+0.1%BSA+10mM HEPES+2mM Glus/ml ammonia benzyls
The μ g/ml of penicillin+100 streptomysins) prepare 18% complement (CEDARLANE).It is prepared for two types：3.3 μ g/ml and 30 μ g/
Ml control antibodies (mouse IgG 1, κ or mouse IgG 2b, κ) and anti-PTPRS antibody.By using the non-radioactives of CytoTox 96
The kit (Promega) of property CTA is measured.As a result, 13G5-52 and 13G5-57 show about 20%
Anti- hPTPRS/CHO target CDC active (Figure 16 A).On the other hand, 13G5-52 and 13G5-57 shows about 100%
The CDC of anti-mPTPRS/CHO target is active (Figure 16 B).
Embodiment 5
The preparation of chimeric antibody
As the hybridoma for producing the anti-PTPRS antibody of mouse, following hybridoma has been used.
Hybridoma 9H5-4 (accession number：FERM BP-11356)
Hybridoma 10F7-38 (accession number：FERM BP-11357)
Hybridoma 13G5-52 (accession number：FERM BP-11358)
Hybridoma 13G5-57 (accession number：FERM BP-11359)
Hybridoma 14A8-85 (accession number：FERM BP-11360)
Hybridoma 22H8-84 (accession number：FERM BP-11361)
Hybridoma 49F2-30 (accession number：FERM BP-11362)
1. the confirmation of the isotype of constant region
Confirm from 7 kinds of hybridomas (9H5-4,10F7-38,13G5-52,13G5-57,14A8-85,22H8-84 and
The isotype of the constant region of each of mouse antibodies caused by 49F2-30).
In order to be confirmed, mouse monoclonal antibody isotype parting kit (catalog number (Cat.No.) has been used：MMT1；Serotec
Product；Oxford, UK) or Pierce Rapid ELISA mouse mAb isotypes parting kits (Thermo Fisher
), and such 9H5-4,10F7-38,13G5-52,13G5-57,14A8-85,22H8-84 and 49F2-30 Scientific
Hybridoma culture supernatants (as sample).
As a result, the isotype of antibody as caused by 13G5-52 and 13G5-57 hybridomas is to include mouse IgG 2b works
For the isotype of heavy chain and κ as light chain.On the other hand, by 9H5-4,10F7-38,14A8-85,22H8-84 and 49F2-30
The isotype of antibody caused by hybridoma is to include mouse IgG 1 as heavy chain and the isotype including κ as light chain.
2. the cDNA of the variable region of the anti-PTPRS antibody of encoding murine clone
2-1) the separation of total serum IgE
By using being obtained commercially kit " RNeasy Mini kits " (Qiagen, catalog number (Cat.No.)：74106), according to examination
Specification appended by agent box, total serum IgE is separated from 7 kinds of hybridomas.By from 5x106Cell number hybridoma
Strain is prepared to obtain about 30 μ g total serum IgE.
2-2) cDNA of encoding murine weight chain variable district amplification and fragmentation
Using 5 μ g in 2-1) in separation total serum IgE, utilize 5'RACE PCR method amplification coding murine heavy chains variable region
cDNA.In amplification, the kit being obtained commercially has been used " to be used for rapid amplifying cDNA END 5'RACE systems (5'RACE
System for Rapid Amplification of cDNA ENDs), 2.0 editions kits " (Invitrogen, catalog number (Cat.No.)：
18374-058).Details as Follows.First, using reverse transcriptase from 2-1) in the total serum IgE that obtains synthesize the first chain cDNA.Now,
Use the antisense primer (GSP1) being illustrated below.
The GSP1 primers for being used for expanding cDNA are used according to the isotype of each murine heavy chain.For example, following antisense draws
Thing be used to clone 9H5-4,10F7-38,14A8-85,22H8-84 and 49F2-30 hybridoma (comprising mouse IgG 1 as weight
Chain) weight chain variable district.
GSP1 primers：mu IgG1VH-GSP1
Sequence：5'-CCA GGA GAG TGG GAG AGG CTC TTC TCA GTA TGG TGG-3'(36-mer)
(SEQ ID NO：39) GSP2 primers：mu IgG1VH-GSP2
Sequence：5'-GGC TCA GGG AAA TAG CCC TTG ACC AGG CAT CC-3'(32-mer)(SEQ ID
NO：40)
Similarly, for example, following antisense primer can be used for clone 9H5-4,10F7-38,14A8-85,22H8-84 and
The weight chain variable district of 49F2-30 hybridomas (comprising mouse IgG 1 as heavy chain).
GSP1 primers：mu IgGHy1-GSP1
Sequence：5'-TCC AGA GTT CCA GGT CAC TGT CAC-3'(24-mer)(SEQ ID NO：11)
GSP2 primers：mu IgG Hγ1-GSP2
Sequence：5'-AGG GGC CAG TGG ATA GAC AGA TGG-3'(32-mer)(SEQ ID NO：13)
And following antisense primer is used to clone 13G5-52 and 13G5-57 hybridomas (comprising mouse IgG 2b as heavy chain)
Weight chain variable district.
GSP1 primers：mu IgGHγ2B-GSP1
Sequence：5'-TCC AGA GTT CCA AGT CAC AGT CAC-3'(24-mer)(SEQ ID NO：41)
GSP2 primers：mu IgG Hγ2B-GSP2
Sequence：5'-AGG GGC CAG TGG ATA GAC TGA TGG-3'(24-mer)(SEQ ID NO：42).
In addition, by using terminal deoxyribotide transferase (TdT) on the first chain cDNA 3'- ends, core is added
Thuja acid homopolymer dC.In addition, use anchor primer (the SEQ ID with the nucleotide polymer complementary with dC (anchor series)
NO：12) with antisense primer (GSP2), cDNA has been expanded using PCR methods.In addition, be template by using the PCR primer of acquisition,
And use AUAP primers (SEQ ID NO：14) with antisense primer (GSP2), cDNA has been expanded using Chao Shi PCR methods.In addition,
The PCR primer has been purified using 1.5% low melting-point agarose method.
5'RACE anchor primer (SEQ ID NO:12)：5'-GGC CAC GCG TCG ACT AGT ACG GGI IGG
GII GGG IIG-3'(36-mer)
AUAP primers (SEQ ID NO for 5'RACE：14)：5'-GGC CAC GCG TCG ACT AGT AC-3'
(20-mer)
2-3) cDNA of encoding murine light chain variable district amplification and fragmentation
By with 2-2) it is similar in a manner of from 2-1) in the cDNA of total serum IgE amplification coding mouse light chain variable region that separates.
Because this 7 kinds of antibody include mouse Ig κ light chains, therefore following antisense primer can be used for cloned light chain.
GSP1 primers：Mu IgVL5RACE-GSP1
Sequence：5'-TTC ACT GCC ATC AAT CTT CCA CTT-3'(24-mer)(SEQ ID NO：15)
GSP2 primers：Mu IgVL5RACE-GSP2
Sequence：5'-GAT GGATAC AGT TGG TGC AGC-3'(21-mer)(SEQ ID NO:16)
Obtained PCR primer is purified using 1.5% low melting-point agarose method.
2-4) the measure in the confirmation of cDNA base sequence and CDR region domain
According to the specification appended by kit, kit " the Zero Blunt TOPO PCR being obtained commercially are used
Cloning kits " (Invitrogen, catalog number (Cat.No.)：1325137) by 2-2) in obtain weight chain variable district and 2-3) in obtain
The cDNA fragments of light chain variable district be each cloned into pCR4Blunt-TOPO carriers, and be introduced into Escherichia coli (E.coli)
Competent cell, to produce Escherichia coli transformant.Plasmid is obtained from the transformant, plasmid DNA samples are delivered into Operon
Biotechnology Co.Ltd (Tokyo) carry out sequence analysis to confirm the cDNA base sequences in plasmid.In order to analyze sequence
Row, " assembling of Sequencher DNA sequence dnas and analysis software 4.2.2 versions (Sequencher DNA sequence are used
Assembly and analysis software version4.2.2) (Gene Codes Corporation) " and
" GENETYX-MAC11.1.1 versions " software (GENETYX CORPORATION) ".
Include frameshit, nonsense mutation for nearby occurring (hereinafter referred to as " CDR region ") by complementary determining region etc. and
Become the RNA of inactivation transformant, be extracted the transformant with correct sequence.In addition, the cDNA alkali being just included in plasmid
Basic sequence is to immunoglobulin database (IgBLAST, URL:Www.ncbi.nlm.nih.gov/igblast/) enter with homology
Confirmation gone to determine the CDR region (CDR in each variable region；CDR1, CDR2, CDR3) sequence, use Kabat numbering system
Unite (Kabat et al., 1991, sequences of Proteins of Immunological Interest, National
Institutes of Health Publication No.91-3242, the 5th edition, United States Department of
Health and Human Services, Bethesda, MD) according to assay framework region and the sequence of variable region.
The nucleotide sequence of the weight chain variable district of the anti-PTPRS mouse 9H5-4 antibody obtained is SEQ ID NO：43, amino
Acid sequence is SEQ ID NO：44.The amino acid sequence of CDR1, CDR2 and CDR3 in the weight chain variable district of mouse 9H5-4 antibody
It is SEQ ID NO respectively：45、SEQ ID NO：46 and SEQ ID NO：47.
The nucleotide sequence of the light chain variable district of the anti-PTPRS mouse 9H5-4 antibody obtained is SEQ ID NO：48, amino
Acid sequence is SEQ ID NO：49.The amino acid sequence of CDR1, CDR2 and CDR3 in the light chain variable district of mouse 9H5-4 antibody
It is SEQ ID NO respectively：50、SEQ ID NO：51 and SEQ ID NO：52.
And the weight chain variable district and light chain variable of anti-the PTPRS mouse 10F7-38 antibody and 14A8-85 antibody of acquisition
The nucleotide sequence in area is identical with those of 9H5-4 antibody, includes CDR1, CDR2 and CDR3 sequence.
The nucleotide sequence of the weight chain variable district of the anti-PTPRS mouse 13G5-57 antibody obtained is SEQ ID NO：53, ammonia
Base acid sequence is SEQ ID NO：54.The amino acid of CDR1, CDR2 and CDR3 in the weight chain variable district of mouse 13G5-57 antibody
Sequence is SEQ ID NO respectively：55、SEQ ID NO：56 and SEQ ID NO：57.
The nucleotide sequence of the light chain variable district of the anti-FTPRS mouse 13G5-57 antibody obtained is SEQ ID NO：58, ammonia
Base acid sequence is SEQ ID NO：59.The amino acid of CDR1, CDR2 and CDR3 in the light chain variable district of mouse 13G5-57 antibody
Sequence is SEQ ID NO respectively：60、SEQ ID NO：61 and SEQ ID NO：62.
And the weight chain variable district of anti-PTPRS mouse 13G5-52 antibody and the nucleotide sequence of light chain variable district of acquisition
It is identical with those of 13G5-57 antibody, include CDR1, CDR2 and CDR3 sequence.
The nucleotide sequence of the weight chain variable district of the anti-PTPRS mouse 22H8-84 antibody obtained is SEQ ID NO：63, ammonia
Base acid sequence is SEQ ID NO：64.The amino acid of CDR1, CDR2 and CDR3 in the weight chain variable district of mouse 22H8-84 antibody
Sequence is respectively SEQ ID NO：65、SEQ ID NO：66 and SEQ ID NO：67.
The nucleotide sequence of the light chain variable district of the anti-PTPRS mouse 22H8-84 antibody obtained is SEQ ID NO：68, ammonia
Base acid sequence is SEQ ID NO：69.The amino acid of CDR1, CDR2 and CDR3 in the light chain variable district of mouse 22H8-84 antibody
Respectively SEQ ID NO：70、SEQ ID NO：71 and SEQ ID NO：72.
The nucleotide sequence of the weight chain variable district of the anti-PTPRS mouse 49F2-30 antibody obtained is SEQ ID NO：25, ammonia
Base acid sequence is SEQ ID NO：26.The amino acid of CDR1, CDR2 and CDR3 in the weight chain variable district of mouse 49F2-30 antibody
Sequence is respectively SEQ ID NO：27、SEQ ID NO：28 and SEQ ID NO：29.
The nucleotide sequence of the light chain variable district of the anti-PTPRS mouse 49F2-30 antibody obtained is SEQ ID NO：30, ammonia
Base acid sequence is SEQ ID NO：31.The amino acid of CDR1, CDR2 and CDR3 in the light chain variable district of mouse 49F2-30 antibody
Sequence is respectively SEQ ID NO：32、SEQ ID NO：33 and SEQ ID NO：34.
The nucleotide sequence (471bp) of the weight chain variable district of the anti-PTPRS mouse 9H5-4 antibody obtained is shown in following (SEQ
ID NO：43).Capitalization shows mouse 9H5-4VH variable regions, and lowercase shows the heavy chain constant region of mouse IgG 1.
ATGGAGTTGGGACTGAGCTGGGTATTTCTTGTGGCTCTTTTGAATGGTGTCCAGTGTCAGGTGCAGCTTGTAGAGAC
CGGGGGAGGCTTGGTGAGGCCTGGAAATTCTCTGAAACTCTCCTGTGTTACCTCGGGATTCACTTTCAGTAACTACC
GGATGCACTGGCTTCGCCAGCCTCCAGGGAAGAGGCTGGAGTGGATTGCTGTAATTACAGTCAAATCTGATAATTAT
GGAGCAAATTATGCAGAGTCTGTGAAAGGCAGATTCACTATTTCAAGAGATGATTCAAAAAGCAGTGTCTACCTGCA
GATGAACAGATTAAGAGAGGAAGACACTGCCACTTATTATTGTAGTAGATCGGTCTACTATGGTTACGTCCTAGCCT
TTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCAgccaaaacgacacccccatctgtctatccactggcc
cctaagggc
The amino acid sequence (157a.a) of the weight chain variable district of mouse 9H5-4 antibody is shown in following (SEQ ID NO：44).
Capitalization shows the sequence of VH variable regions, and lowercase shows the heavy chain constant region of mouse IgG 1.It is subject to the part meaning of underscore
Refer to signal sequence, the part for being subject to double underline means CDR region domain (CDR1, CDR2, CDR3).
MELGLSWVFLVALLNGVQCQVQLVETGGGLVRPGNSLKLSCVTSGFTFS
WLRQPPGKRLEWIARFTISRDDSKSSVYLQMNRLREEDTATYYCSRSVYYGYVL
AFDYWGQGTTLTVSSakttppsvyplapkg
The CDR1 of the weight chain variable district of 9H5-4 antibody is NYRMH (SEQ ID NO：45), the weight chain variable of 9H5-4 antibody
The CDR2 in area is VITVKSDNYGANYAESVKG (SEQ ID NO：46), the weight chain variable district CDR3 of 9H5-4 antibody is
SVYYGYVLAFDY(SEQ ID NO：47).
The nucleotide sequence (402bp) of the light chain variable district of the anti-PTPRS mouse 9H5-4 antibody obtained is shown in following (SEQ
ID NO：48).Capitalization shows mouse 9H5-4VH variable regions, and lowercase shows mouse Ig κ constant region of light chain.
ATGATGTCCTCTGCTCAGTTCCTTGGTCTCCTGTTGCTCTGTTTTCAAGGTACCAGATGTGATATCCAGATGACACA
GACTACATCCTCCCTGTCTGCCTCTCTGGG AGACAGAGTCACCATCAGTTGCAGGGCAAGTCAGGACATTAGCAAT
TATTTAAACTGGTATCAGCAGAAACCAGATGGAACTGTTAAACTCCTGATCTACTACACATCAAGATTACACTCAGG
AGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGAACAGATTATTCTCTCACCATTAGCAACCTGGAGCAAGAAGATA
TTGCCACTTACTTTTGCCAACAGGGTAATACGCTTCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAAcgg
gctgatgctgcaccaact
The amino acid sequence (134a.a) of the light chain variable district of mouse 9H5-4 antibody is shown in following (SEQ ID NO：49).
Capitalization shows the sequence of mouse 9H5-4VH variable regions, and lowercase shows mouse Ig κ constant region of light chain.It is subject to underscore
Part mean signal sequence, the part for being subject to double underline means CDR region (CDR1, CDR2, CDR3).
MMSSAQFLGLLLLCFQGTRCDIQMTQTTSSLSASLGDRVTISC
WYQQKPDGTVKLLIYGVPSRFSGSGSGTDYSLTISNLEQEDIATYFC
WTFGGGTKLEIKradaapt
The CDR1 of the light chain variable district of 9H5-4 antibody is RASQDISNYLN (SEQ ID NO：50), 9H5-4 antibody is light
The CDR2 of chain variable region is YTSRLHS (SEQ ID NO：51), the CDR3 of the light chain variable district of 9H5-4 antibody is QQGNTLP
(SEQ ID NO：52).
The nucleotide sequence (465bp) of the weight chain variable district of anti-PTPRS mouse 13G5-57 antibody is shown in following (SEQ ID
NO：53).Capitalization shows mouse 13G5-57VH variable regions, and lowercase shows mouse IgG 2b heavy chain constant region.
ATGAACTTGGGGCTCAGCTTGATTTTCCTTGTCCTTGTTTTAAAAGGTGTCCAGTGTGAAGTGAAGCTGGTGGAGTC
TGGGGGAGGCTTAGTGCAGCCTGGAGGGTCCCTGAAACTCTCCTGTGCAACCTCTGGATTCACTTTCAGTGACTATT
ACATGTATTGGGTTCGCCAGACTCCAGAGAAGAGGCTGGAGTGGGTCGCATACATTAGTAATGGTGGTGGTAGCACC
TATTATCCAGACACTGTAAAGGGCCGATTCACCATCTCCAGAGACAATGCCAAGAACACCCTGTACCTGCAAATGAG
CCGTCTGAAGTCTGAGGACACAGCCATGTATTACTGTGCAAGACATGTTTACTACGGGAGGAACTATGCTATGGACT
ACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCAgccaaaacaacacccccatcagtctatccactggcccctaag
ggc
The amino acid sequence (155a.a) of the weight chain variable district of mouse 13G5-57 antibody is shown in following (SEQ ID NO：
54).Capitalization shows the sequence of VH variable regions, and lowercase shows mouse IgG 2b heavy chain constant region.It is subject to the portion of underscore
Divide and mean signal sequence, the part for being subject to double underline means CDR region domain (CDR1, CDR2, CDR3).
MNLGLSLIFLVLVLKGVQCEVKLVESGGGLVQPGGSLKLSCATSGFTFS
WVRQTPEKRLEWVARFTISRDNAKNTLYLQMSRLKSEDTAMYYCARWGQGTSVTVSSakttppsvyplapkg
The CDR1 of the weight chain variable district of 13G5-57 antibody is DYYMY (SEQ ID NO：55), the heavy chain of 13G5-57 antibody
The CDR2 of variable region is YISNGGGSTYYPDTVKG (SEQ ID NO：56), the CDR3 of the weight chain variable district of 13G5-57 antibody is
HVYYGRNYAMDY(SEQ ID NO：57).
The light chain variable district nucleotide sequence (465bp) of the anti-PTPRS mouse 13G5-57 antibody obtained is shown in following (SEQ
ID NO：58).Capitalization shows mouse 13G5-57VH variable regions, and lowercase shows mouse Ig κ constant region of light chain.
ATGAACTTGGGGCTCAGCTTGATTTTCCTTGTCCTTGTTTTAAAAGGTGTCCAGTGTG
AAGTGAAGCTGGTGGAGTCTGGGGGAGGCTTAGTGCAGCCTGGAGGGTCCCTGAAACTCTCCTGTGCAACCTCTGGA
TTCACTTTCAGTGACTATTACATGTATTGGGTTCGCCAGACTCCAGAGAAGAGGCTGGAGTGGGTCGCATACATTAG
TAATGGTGGTGGTAGCACCTATTATCCAGACACTGTAAAGGGCCGATTCAC
CATCTCCAGAGACAATGCCAAGAACACCCTGTACCTGCAAATGAGCCGTCTGAAGTCTGAGGACACAGCCATGTATT
ACTGTGCAAGACATGTTTACTACGGGAGGAACTATGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCC
TCAgccaaaacaacacccccatcagtctatccactggcccctaagggc
The amino acid sequence (155a.a) of the light chain variable district of mouse 13G5-57 antibody is shown in following (SEQ ID NO：
59).Capitalization shows the sequence of mouse 13G5-57VH variable regions, and lowercase shows mouse Ig κ constant region of light chain.It is subject to
Underscore part means signal sequence, and the part for being subject to double underline means CDR region domain (CDR1, CDR2, CDR3).
MMSSAQFLGLLLLCFQGTRCDIQMTQTTSSLSASLGDRVTISC
WYQQKPDGTVKLLIYGVPSRFSGSGSGTDYSLTISNLEQEDIATYFC
TFGGGTKLEIKradaaptvsifppsseqltsggasvvcf
The CDR1 of the light chain variable district of 13G5-57 antibody is RASQDISNYLN (SEQ ID NO：60), 13G5-57 antibody
The CDR2 of light chain variable district be YTSRLHS (SEQ ID NO：61), the CDR3 of the light chain variable district of 13G5-57 antibody is
QQGNTLPY(SEQ ID NO：62).
The nucleotide sequence (458bp) of the weight chain variable district of anti-PTPRS mouse 22H8-84 antibody is shown in following (SEQ ID
NO：63).Capitalization shows mouse 22H8-84VH variable regions, and lowercase shows the heavy chain constant region of mouse IgG 1.
ATGGAATGTAACTGGATACTTCCTTTTATTCTGTCAGTAACTTCAGGTGTCTACTCACAGGTTCAGCTCCAGCAGTC
TGGGGCTGAGCTGGCAAGACCTGGGGCTTCAGTGAAGTTGTCCTGCAAGGCTTCTGGCTACACCTTTACTAGCTACT
GGATGCAGTGGGTAAAACAGAGGCCTGGACAGGGTCTGGAATGGATTGGGGCTATTTATCCTGGAGATGGTGATACT
AGGTACACTCAGAAGTTCAAGGGCAAGGCCACATTGACTGCAGATAAATCCTCCAGCACAGCCTACATGCAACTCAG
CAGCTTGGCATCTGAGGACTCTGCGGTCTATTACTGTGCAAGAAGGATTTACTACGGCTATTACTATGCTATGGACT
ACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCagccaaaacgacacccccatctgtctatccactggcccc
The amino acid sequence (152a.a) of the weight chain variable district of mouse 22H8-84 antibody is shown in following (SEQ ID NO：
64).Capitalization shows the sequence of VH variable regions, and lowercase shows the heavy chain constant region of mouse IgG 1.It is subject to the portion of underscore
Divide and mean signal sequence, the part for being subject to double underline means CDR region (CDR1, CDR2, CDR3).
MECNWILPFILSVTSGVYSQVQLQQSGAELARPGASVKLSCKASGYTFT
WVKQRPGQGLEWIGKATLTADKSSSTAYMQLS SLASEDSAV YYCARWGQGTSVTVSSakttppsvypla
The CDR1 of the weight chain variable district of 22H8-84 antibody is SYWMQ (SEQ ID NO：65), the heavy chain of 22H8-84 antibody
The CDR2 of variable region is AIYPGDGDTRYTQKFKG (SEQ ID NO：66), the CDR3 of the weight chain variable district of 22H8-84 antibody is
RIYYGYYYAMDY(SEQ ID NO：67).
The nucleotide sequence (430bp) of the light chain variable district of the anti-PTPRS mouse 22H8-84 antibody obtained is shown in following
(SEQ ID NO：68).Capitalization shows mouse 22H8-84VH variable regions, and lowercase shows mouse Ig κ constant region of light chain.
ATGGAGACAGACACAATCCTGCTATGGGTGCTGCTGCTCTGGGTTCCAGGCTCCACTGGTGACATTGTGCTGACCCA
ATCTCCAGCTTCTTTGGCTGTGTCTCTAGGGCAGAGGGCCACCATCTCCTGCAAGGCCAGCCAAAGTGTTGATTATG
ATGGTGATAGTTATATGAACTGGTACCAACAGAAACCAGGACAGCCACCCAAACTCCTCATCTATGCTGCATCCAAT
CTAGAATCTGGGATCCCAGCCAGGTTTAGTGGCAGTGGGTCTGGGACAGACTTCACCCTCAACATCCATCCTGTGGA
GGAGGAGGATGCTGCAACCTATTACTGTCAGCAAAGTAATGAGGATCCTCTCACGTTCGGTGCTGGGACCAAGCTGG
AGCTGAAAcgggctgatgctgcaccaactgtatccatcaagggcg
The amino acid sequence (143a.a) of the light chain variable district of mouse 22H8-84 antibody is shown in following (SEQ ID NO：
69).Capitalization shows the sequence of mouse 22H8-84VH variable regions, and lowercase shows mouse Ig κ constant region of light chain.It is subject to
The part of underscore means signal sequence, and the part for being subject to double underline means CDR region domain (CDR1, CDR2, CDR3).
METDTILLWVLLLWVPGSTGDIVLTQSPASLAVSLGQRATISC
WYQQKPGQPPKLLIYGIPARFSGSGSGTDFTLNIHPVEEEDAATYYC
TFGAGTKLELKradaaptvsikg
The CDR1 of the light chain variable district of 22H8-84 antibody is KASQSVDYDGDSYMN (SEQ ID NO：70), 22H8-84
The CDR2 of the light chain variable district of antibody is AASNLES (SEQ ID NO：71), the CDR3 of the light chain variable district of 22H8-84 antibody is
QQSNEDPL(SEQ ID NO：72).
The nucleotide sequence (469bp) of the weight chain variable district of anti-PTPRS mouse 49F2-30 antibody is shown in following (SEQ ID
NO：25).Capitalization shows mouse 49F2-30VH variable region, and lowercase shows the heavy chain constant region of mouse IgG 1.
ATGAACTTCGGGCTCAGGTTGATTTTCCTTGCCCTCATTTTAAAAGGTGTCCAGTGTGAGGTGCAGCTGGTGGAGTC
TGGGGGAGACTTAGTGAAGCCTGGAGGGTCCCTGAAACTCTCCTGTGCAGCCTCTGGATTCATTTTCAGTAGCTATG
GCATGTCTTGGGTTCGCCAGACTCCAGACAAGAGGCTGGAGTGGGTCGCAACCATTAGTAGTGGTGGTAGTGACACC
TATTATCCAGACAGTGTGAAGGGGCGATTCACCATCTCCAGAGACAATGCCAACAACACCCTGTACCTGCAAATGAG
CAGTCTGAAGTCTGAGGACACAGCCATGTATTACTGTGCAAGACAGGTCTACTATGGTCTTTACTGGTATTTCGATG
TCTGGGGCGCAGGGACCACGGTCACCGTCTCCTCAgccaaaacgacacccccatctgtctatccactggcccctaag
ggcgaat
The heavy chain variable amino acid sequence (156a.a) of mouse 49F2-30 antibody is shown in following (SEQ ID NO：26).
Capitalization shows VH variable genes, and lowercase shows the heavy chain constant region of mouse IgG 1.It is subject to the sequence display letter of underscore
Number sequence, the sequence for being subject to double underline show CDR region (CDR1, CDR2, CDR3).
MNFGLRLIFLALILKGVQCEVQLVESGGDLVKPGGSLKLSCAASGFIFSWVRQTPDKRLEWVA
RFTISRDNANNTLYLQMSSLKSEDTAMYYCARWGAGTTVTVSS
akttppsvyplapkge
The CDR1 of the weight chain variable district of 49F2-30 antibody is SYGMS (SEQ ID NO：27), the heavy chain of 49F2-30 antibody
The CDR2 of variable region is TISSGGSDTYYPDSVKG (SEQ ID NO：28), the CDR3 of the weight chain variable district of 49F2-30 antibody is
QVYYGLYWYFDV(SEQ ID NO：29).
The nucleotide sequence (413bp) of the light chain variable district of the anti-PTPRS mouse 49F2-30 antibody obtained is shown in following
(SEQ ID NO：30).Capitalization shows mouse 49F2-30VL variable region, and lowercase shows mouse Ig κ chain constants
Area.
ATGGAGTCACAGATTCAGGTCTTTGTATTCGTGTTTCTCTGGTTGTCTGGTGTTGACGGAGACATTGTGATGACCCA
GTCTCACAAATTCATGTCCACATCAGTAGGAGACAGGGTCAGCATCATTTGTAAGGCCAGTCAGGATGTGAATACTG
CTGTAGCCTGGTATCAACAGAAACCAGGACAATCTCCTAAATTACTGATTTACTCGGCATCCTACCGGTACACTGGA
GTCCCTGATCGCTTCACTGGCAGTGGATCTGGGACGGATTTCACTTTCACCATCAGCAGTGTGCAGGCTGAAGACCT
GGCAATTTATTACTGTCAGCAACATTATAGTACTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAAcggg
ctgatgctgcaccaactgtatccatcaa
The amino acid sequence (137a.a) of the light chain variable district of mouse 49F2-30 antibody is shown in following (SEQ ID NO：
31).Capitalization shows that mouse 49F2-30VL variable regions and lowercase show mouse Ig κ constant region of light chain.It is subject to underscore
Sequence show signal sequence, be subject to double underline sequence show CDR region (CDR1, CDR2, CDR3).
MESQIQVFVFVFLWLSGVDGDIVMTQSHKFMSTSVGDRVSIICWYQQKPGQSPKLLIYGVPDRFTGSGSGTDFTFTISSVQAEDLAIYYCYTFGGGTKLEIK radaaptvsi
The CDR1 of the light chain variable district of 49F2-30 antibody is KASQDVNTAVA (SEQ ID NO：32), 49F2-30 antibody
The CDR2 of light chain variable district be SASYRYT (SEQ ID NO：33), the CDR3 of the light chain variable district of 49F2-30 antibody is
QQHYSTP(SEQ ID NO：34).
3. the preparation of the expression vector of chimeric antibody
3-1) the cDNA of encoding human Ig constant regions clone
According to the specification appended by kit, kit " the Zero Blunt TOPO PCR being obtained commercially are used
Cloning kits " (Invitrogen, catalog number (Cat.No.)：1325137) from the total serum IgE human cloning IgG1 heavy chain constant region of human PBMC
With the cDNA of people's Ig κ constant region of light chain, it is each cloned into pCR4Blunt-TOPO carriers, and introduce E. coli competent
Cell, to produce Escherichia coli transformant.Above-mentioned plasmid is obtained from the transformant, plasmid DNA samples are delivered into Operon
Biotechnology Co., Ltd.s (Tokyo) carry out sequence analysis to confirm the cDNA base sequences in plasmid.
3-2) it is fitted together to the preparation of the expression vector of 9H5-4 (10F7-38,14A8-85) antibody
In order to prepare the cDNA of the heavy chain of encoding chimera PTPRS antibody, by the mouse 9H5-4 antibody obtained in 2-2
Weight chain variable district with by human IgG heavy chain constant region be integrated into pEE6.4 carriers therein (Lonza Biologies, Slough,
UK) merge, the weight chain variable district of mouse 9H5-4 antibody is expanded by PCR methods, obtain the PCR of the length with about 450 bases
Product.Now, primer is following primer.The PCR primer obtained using 1.5% low melting-point agarose method purifying.
For the primer for the heavy chain for expressing chimeric 9H5-4 antibody
1) forward primer：chi10F7VH-IF(Hind3)
Sequence：5'ttt AAG CTT gcc gcc acc ATG GAG TTG GGA CTG AGC TGG3'(39-mer)
(SEQ ID NO：73)
2) reverse primer：chi10F7VH-462R(ApaI)
Sequence：5'cga tgg gcc ctt ggt get age TGA GGA GAC TGT GAG AGT GGT3'(42-
mer)(SEQ ID NO：74)
The mouse 9H5-4 heavy chain of antibody variable region obtained by PCR methods from 2-2 obtains " coding 9H5-4 weight chain variables
The PCR primer in area ".The PCR primer of 9H5-4 weight chain variable districts is encoded using Hind III and Apa I digestion with restriction enzyme,
And purified using 1.5% Ago-Gel method.It is dissolved in ddH2O is to provide the cDNA fragments of encoding heavy chain variable region
Solution.
By using primer chi10F7VH-IF (Hind3) and chi10F7VH-462R (Apal) (using Hind III
The preferred limit that will be used to be cloned into pEE6.4 carriers (Lonza Biologies, Slough, UK) as cloning site with Apal
Property site (Hind III and Apal) processed and preferable Kozak sequences (GCCGCCACC) introduce the primer), by PCR from
PCR4Blunt-TOPO plasmid clonings (include 9H5-4 VHCode area) amplification obtain cDNA 9H5-4 VHCode area.
Chi9H5-4VH-pEE6.4 carriers include the heavy chain constant region of human IgG1.Using Hind III and Apal by VHPCR fragment frame
In interior insertion pEE6.4 carriers.Construct is have studied using cDNA base sequence analysis, plasmid DNA samples are delivered into Operon
Biotechnology Co., Ltd. (Tokyo) carry out sequence analysis to confirm the cDNA base sequences in plasmid.
In order to prepare the cDNA of the light chain of encoding chimera 9H5-4 antibody, using based on overlap-extension PCR (overlap
Extension) PCR technology, the mouse 9H5-4 antibody light chains variable region obtained in 2-3 has been merged therefrom with being obtained in 3-2
People's Ig κ constant region of light chain PCR fragment amplification length be about 730 bases PCR primer.
The PCR primer of coding 9H5-4 light chain variable districts is digested using Hind III and EcoRI restriction endonuclease,
And purified using 1.5% Ago-Gel method.It is dissolved in ddH2O is to provide the cDNA fragments of coding light chain variable region
Solution.
(clone will be used to enter by using primer chi11G9VL-IF (Hind) and chi11G9VL-726R (RI)
The preferred restriction site (Hind III and EcoRI) of pEE14.4 carriers (Lonza Biologies) and preferable Kozak sequences
Row introduce the primer), 9H5-4 V (is included from pCR4Blunt-TOPO plasmid clonings by PCRLCode area) expand institute
The 9H5-4 of acquisition VLCode cDNA.Chi9H5-4VL-pEE14.4 carriers include κ constant region of light chain.Using Hind III and
EcoRI is by VLPCR fragment inframe inserts pEE14.4 carriers.Construct have studied by cDNA base sequence analysis.
For the primer for the light chain for expressing chimeric 9H5-4 antibody
1) forward primer：chi11G9VL-IF(Hind)
Sequence：5'acc AAG CTT gcc gcc acc ATG ATG TCC TCT GCT CAG TTC3'(39-mer)
(SEQ ID NO：75)
2) reverse primer：chi11G9VL-408R
Sequence：5'agc cac agt tcg TTT GAT TTC CAG CTT GGT GCC 3'(33-mer)(SEQ ID
NO：76)
3) forward primer：chi11G9VL-385F
Sequence：5'CTG GAAATC AAA cga act gtg gct gca cca tct 3"(33-mer)(SEQ ID
NO：77)
4) reverse primer：chi11G9VL-726R(RI)
Sequence：5'aaa GAA TTC cta gca ctc tcc cct gtt gaa3'(30-mer)(SEQ ID NO：
78)
3-2) it is fitted together to the preparation of the expression vector of 13G5-57 (13G5-52) antibody
In order to prepare the cDNA of the heavy chain of encoding chimera PTPRS antibody, the mouse 13G5-57 antibody that will have been obtained in 2-2
Weight chain variable district with by human IgG heavy chain constant region be integrated into pEE6.4 carriers therein (Lonza Biologics,
Slough, UK) fusion.In a manner of similar to 9H5-4 antibody, acquisition and purified pcr product.Now, introducing is following primer.
For the primer for the heavy chain for expressing chimeric 13G5-57 antibody
1) forward primer：chi13G5.57VH-IF(Hind3)
Sequence：5'ttt AAG CTT gcc gcc acc ATG AAC TTG GGG CTC AGC TTG3'(39-mer)
(SEQ ID NO：79)
2) reverse primer：chi13G5.57VH-456R(ApaI)
Sequence：5'cga tgg gcc ctt ggt gct agc TGA GGA GAC GGT GAC TGA GGT3'(42-
mer)(SEQ ID NO：80)
In order to prepare the cDNA of the light chain of encoding chimera 13G5-57 antibody, using based on the technology of Overlap extension PCR from its
In merged the mouse 13G5-57 antibody light chains variable region that is obtained in 2-3 and the PCR of the people's Ig κ constant region of light chain obtained in 3-2
Fragment amplification length is the PCR primer of about 730 bases.
For the primer for the light chain for expressing chimeric 13G5-57 antibody
1) forward primer：chi11G9VL-IF(Hind)
Sequence：5'acc AAG CTT gcc gcc acc ATG ATG TCC TCT GCT CAG TTC3'(39-mer)
(SEQ ID NO：81)
2) reverse primer：chi11G9VL-408R
Sequence：5'agc cac agt tcg TTT GAT TTC CAG CTT GGT GCC 3'(33-mer)(SEQ ID
NO：82)
3) forward primer：chi11G9VL-385F
Sequence：5'CTG GAAATC AAA cga act gtg gct gca cca tct 3'(33-mer)(SEQ ID
NO：83)
4) reverse primer：chi11G9VL-726R(RI)
Sequence：5'aaa GAA TTC cta gca ctc tcc cct gtt gaa3'(30-mer)(SEQ ID NO：
84)
In a manner of the expression vector of 9H5-4 antibody chimeric with preparing is similar, the such of chimeric 13G5-57 antibody is prepared
The expression vector of heavy chain and light chain.
3-2) prepare the expression vector of chimeric 22H8-84 antibody
In order to prepare the cDNA of the heavy chain of encoding chimera PTPRS antibody, the mouse 22H8-84 antibody that will have been obtained in 2-2
Weight chain variable district with by human IgG heavy chain constant region be integrated into pEE6.4 carriers therein (Lonza Biologics,
Slough, UK) fusion.In a manner of similar to 9H5-4 antibody, acquisition and purified pcr product.Now, primer is to draw as follows
Thing.
For the primer for the heavy chain for expressing chimeric 22H8-84 antibody
1) forward primer：chi22H8VH-IF(Hind3)
Sequence：5'ttt AAG CTT gcc gcc acc ATG GAA TGT AAC TGG ATA CTT3*(39-mer)
(SEQ ID NO：85)
2) reverse primer：chi22H8VH-456R(ApaI)
Sequence：5'cga tgg gcc ctt ggt gct agc TGA GGA GAC GGT GAC TGA GGT3'(42-
mer)(SEQ ID NO：86)
In order to prepare the cDNA of the light chain of encoding chimera 22H8-84 antibody, using based on the technology of Overlap extension PCR from its
In merged the mouse 22H8-84 antibody light chains variable region that is obtained in 2-3 and the PCR of the people's Ig κ constant region of light chain obtained in 3-2
Fragment amplification length is the PCR primer of about 730 bases.
For the primer for the light chain for expressing chimeric 22H8-84 antibody
1) forward primer：chi22H8VL-IF(Hind)
Sequence：5'acc AAG CTT gcc gcc acc ATG GAG ACA GAC ACA ATC CTG3'(39-mer)
(SEQ ID NO：87)
2) reverse primer：chi22H8VL-420R
Sequence：5'agc cac agt tcg TTT CAG CTC CAG CTT GGT CCC 3'(33-mer)(SEQ ID
NO：88)
3) forward primer：chi22H8VL-397F
Sequence：5'CTG GAG CTG AAA cga act gtg gct gca cca tct 3'(33-mer)(SEQ ID
NO：89)
4) reverse primer：chi49F2VL-726R(RI)
Sequence：5'aaa GAA TTC cta gca ctc tcc cct gtt gaa3'(30-mer)(SEQ ID NO：
90)
In a manner of the expression vector of 9H5-4 antibody chimeric with preparing is similar, the such of chimeric 22H8-84 antibody is prepared
The expression vector of heavy chain and light chain.
3-2) the cDNA of the heavy chain of encoding chimera PTPRS antibody preparation
In order to prepare the cDNA of the heavy chain of encoding chimera PTPRS antibody, using the method based on Overlap extension PCR method at it
In merged the mouse 49F2-30 antibody that is obtained in 2-2 weight chain variable district and 3-1 in the human IgG heavy chain constant region that obtains
Change two PCR fragments in PCR fragment, allow double-fiber silk molecule (double filament by the use of the result as crossover operation
Molecule) the method amplification length that part is formed is the PCR primer of 1434 bases.Now, primer (SEQ ID NOS：17
To the 24) primer to be shown in table 1.Obtained PCR primer is purified using 1.5% low melting-point agarose method.
Table 1
In the presence of the human IgG1 obtained in the mouse 49F2-30 heavy chain of antibody variable region and 3-1 obtained in wherein cDNA and 2-2
The region of light chain constant area overlapping.Therefore, by using the region, " coding 49F2-30 is obtained using Overlap extension PCR method
The PCR primer of weight chain variable district ".Coding 49F2-30 heavy chains are digested using Hind III and EcoR I restriction endonuclease
The PCR primer of variable region, and purified using 1.5% Ago-Gel method.It is dissolved in ddH2O is to provide encoding heavy chain
The solution of the cDNA fragments of variable region.
By using primer chi49F2VH-IF (Hind3) and chi49F2VH-1434R (RI) (using Hind III and
The preferred limit that EcoRI will be used to be cloned into pEE6.4 carriers (Lonza Biologics, Slough, UK) as cloning site
Property site (Hind III and EcoRI) processed and preferable Kozak sequences (GCCGCCACC) introduce the primer), by PCR from
PCR4Blunt-TOPO plasmid clonings (include 49F2-30 VHCode area) amplification obtain cDNA 49F2-30 VHCoding
Area.Chi49F2VH-pEE6.4 carriers include the heavy chain constant region of human IgG1.Using Hind III and EcoRI by VHPCR pieces
Section inframe insertion pEE6.4 carriers.Construct is studied using cDNA base sequence analysis.
3-3) the cDNA of the light chain of encoding chimera PTPRS antibody preparation
In order to prepare the cDNA of the light chain of encoding chimera PTPRS antibody, using the technology based on Overlap extension PCR therefrom
The mouse 49F2-30 antibody light chains variable region obtained in 2-3 and the PCR pieces of the people's Ig κ constant region of light chain obtained in 3-2 are merged
Section amplification length is the PCR primer of 726 bases.
The PCR that coding 49F2-30 light chain variable districts are digested using Hind III and EcoR I restriction endonuclease is produced
Thing, and purified using 1.5% Ago-Gel method.It is dissolved in ddH2O is to provide the cDNA pieces of coding light chain variable region
The solution of section.
(it will be used to be cloned into by using primer chi49F2VL-IF (Hind) and chi49F2VL-726R (RI)
Preferred restriction site (Hind III and EcoRI) and preferable Kozak in pEE14.4 carriers (Lonza Biologics)
Sequence introduces the primer), 49F2-30 V (is included from pCR4Blunt-TOPO plasmid clonings by PCRLArea) expand and obtained
The 49F2-30 obtained VLCode cDNA.Chi49F2VL-pEE14.4 carriers include κ constant region of light chain.Using Hind III and
EcoRI is by VLPCR fragment inframe inserts pEE14.4 carriers.Construct is studied using cDNA base sequence analysis.
3-4) it is fitted together to the structure of the dual-gene Lonza expression vectors of PTPRS antibody
Chimeric PTPRS antibody (dual-gene) Lonza expression vectors are built using Standard cloning techniques, wherein will be chimeric
The heavy chain expression vector of PTPRS antibody is combined in a dual-gene carrier with the light chain expression vector of chimeric PTPRS antibody.
Transient expression in 4.HEK-293F cells
Following transient expression vector DNA (80 μ g) is used.
1) chi9H5-4VH/VL DG Lonza carrier DNAs
2) chi3G5-57VH/VL DG Lonza carrier DNAs
3) chi22H8-84VH/VL DG Lonza carrier DNAs
4) chi49F2-30VH/VL DG Lonza carrier DNAs
In the day before transfection, by 293F cells in 250mL conical flask (Corning#431144) with 8x105It is individual thin
Born of the same parents/mL is adjusted to 80mL, in 37 DEG C and 8% CO2Shaken cultivation 7 days under conditions of concentration.
After culture 7 days, the nutrient solution that will undergo the 293F cells of transfection is collected in 50mL test tubes, in 2070g and 4
Centrifuged 5 minutes under conditions of DEG C.Utilize the syringe type filter (catalog number (Cat.No.) 431220 with 0.45 μm of aperture；CORNING)
Filtering supernatant, culture supernatant is gathered together.
5. the purifying of anti-PTPRS chimeric antibodies
Chimeric 9H5-4,13G5-57,22H8-84 and 49F2-30 antibody is purified using protein A affinity chromatography.It will be obtained in 4
Rough antibody liquid each with albumin A affinity column (rProtein A Sepharose Fast Flow (catalog number (Cat.No.) 17-1279-
01；Lot.311272；GE Healthcare) purified.Column condition is as follows.By using combination buffer (20mM phosphoric acid
Sodium, 0.15M NaCl, pH 7.4) and elution buffer (0.1M glycerine-HCl, pH 2.7) progress affinity purification.By in addition
The pH of the fraction of elution is adjusted to about 7.2 with buffer solution (1M Tris-HCl pH 9.5).In order to replace the anti-of purifying with PBS
The buffer solution of body, buffer solution is replaced by using Slide-A-Lyzer MINI Dialysis unit 10kMWCO.
By measuring absorbance at 280nm and 1mg/l being defined as into 1.38OD to calculate the concentration of the antibody of purifying.
Using SDS-PAGE and flow cytometry analysis purifying anti-PTPRS chimeric antibodies (ch9H5-4Ab,
Ch13G5-57Ab, ch22H8-84Ab and ch49F2-30Ab).
The heavy chain of chimeric 9H5-4 antibody and the nucleotide sequence and amino acid sequence of light chain prepared is compiled with one sequence respectively
Number represent.
The nucleotide sequence (1419bp) that anti-PTPRS is fitted together to the heavy chain of 9H5-4 antibody is shown in following (SEQ ID NO：91).
Capitalization shows chimeric 9H5-4VH variable regions, and lowercase shows human IgG1's heavy chain constant region.
ATGGAGTTGGGACTGAGCTGGGTATTTCTTGTGGCTCTTTTGAATGGTGTCCAGTGTCAGGTGCAGCTTGTAGAGAC
CGGGGGAGGCTTGGTGAGGCCTGGAAATTCTCTGAAACTCTCCTGTGTTACCTCGGGATTCACTTTCAGTAACTACC
GGATGCACTGGCTTCGCCAGCCTCCAGGGAAGAGGCTGGAGTGGATTGCTGTAATTACAGTCAAATCTGATAATTAT
GGAGCAAATTATGCAGAGTCTGTGAAAGGCAGATTCACTATTTCAAGAGATGATTCAAAAAGCAGTGTCTACCTGCA
GATGAACAGATTAAGAGAGGAAGACACTGCCACTTATTATTGTAGTAGATCGGTCTACTATGGTTACGTCCTAGCCT
TTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCAgctagcaccaagggcccatcggtcttccccctggca
ccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgac
ggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctact
ccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagccc
agcaacaccaaggtggacaagaaagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacc
tgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctg
aggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggag
gtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcct
gcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaa
ccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaag
aaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggca
gccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccg
tggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacg
cagaagagcctctccctgtctccgggtaaatga
The heavy chain amino acid sequence (472a.a.) that anti-PTPRS is fitted together to 9H5-4 antibody is shown in following (SEQ ID NO：92).
Capitalization shows chimeric 9H5-4VH variable regions, and lowercase shows human IgG1's heavy chain constant region.
MELGLSWVFLVALLNGVQCQVQLVETGGGLVRPGNSLKLSCVTSGFTFSNYRMHWLRQPPGKRLEWIAVITVKSDNY
GANYAESVKGRFTISRDDSKSSVYLQMNRLREEDTATYYCSRSVYYGYVLAFDYWGQGTTLTVSSastkgpsvfpla
psskstsggtaalgclvkdyfpepvtvswnsgaltsgvhtfpavlqssglyslssvvtvpssslgtqtyicnvnhkp
sntkvdkkvepkscdkthtcppcpapellggpsvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgve
vhnaktkpreeqynstyrvvsvltvlhqdwlngkeykckvsnkalpapiektiskakgqprepqvytlppsrdeltk
nqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhyt
qkslslspgk
The light chain nucleic acid sequence (705bp) that anti-PTPRS is fitted together to 9H5-4 antibody is shown in following (SEQ ID NO：93).Capitalization
The chimeric 9H5-4VL variable regions of letter display, lowercase show people's Ig κ constant region of light chain.
ATGATGTCCTCTGCTCAGTTCCTTGGTCTCCTGTTGCTCTGTTTTCAAGGTACCAGATGTGATATCCAGATGACACA
GACTACATCCTCCCTGTCTGCCTCTCTGGGAGACAGAGTCACCATCAGTTGCAGGGCAAGTCAGGACATTAGCAATT
ATTTAAACTGGTATCAGCAGAAACCAGATGGAACTGTTAAACTCCTGATCTACTACACATCAAGATTACACTCAGGA
GTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGAACAGATTATTCTCTCACCATTAGCAACCTGGAGCAAGAAGATAT
TGCCACTTACTTTTGCCAACAGGGTAATACGCTTCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAAcgaa
ctgtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgc
ctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactccca
ggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagact
acgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacagg
ggagagtgctag
The light-chain amino acid sequence (234a.a.) that anti-PTPRS is fitted together to 9H5-4 antibody is shown in following (SEQ ID NO：94).
Capitalization shows chimeric 9H5-4VL variable regions, and lowercase shows people's Ig κ constant region of light chain.
MMSSAQFLGLLLLCFQGTRCDIQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVKLLIYYTSRLHSG
VPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFGGGTKLEIKrtvaapsvfifppsdeqlksgtasvvc
llnnfypreakvqwkvdnalqsgnsqesvteqdskdstyslsstltlskadyekhkvyacevthqglsspvtksfnr
gec
<13G5-57>
The heavy chain of the chimeric 13G5-57 antibody prepared and the nucleotide sequence and amino acid sequence of light chain are respectively by one sequence
Numbering represents.
The heavy chain nucleic acid sequence (1413bp) that anti-PTPRS is fitted together to 13G5-57 antibody is shown in following (SEQ ID NO：95).
Capitalization shows chimeric 13G5-57VH variable regions, and lowercase shows human IgG1's heavy chain constant region.
ATGAACTTGGGGCTCAGCTTGATTTTCCTTGTCCTTGTTTTAAAAGGTGTCCAGTGTGAAGTGAAGCTGGTGGAGTC
TGGGGGAGGCTTAGTGCAGCCTGGAGGGTCCCTGAAACTCTCCTGTGCAACCTCTGGATTCACTTTCAGTGACTATT
ACATGTATTGGGTTCGCCAGACTCCAGAGAAGAGGCTGGAGTGGGTC
GCATACATTAGTAATGGTGGTGGTAGCACCTATTATCCAGACACTGTAAAGGGCCGATTCACCATCTCCAGAGACAA
TGCCAAGAACACCCTGTACCTGCAAATGAGCCGTCTGAAGTCTGAGGACACAGCCATGTATTACTGTGCAAGACATG
TTTACTACGGGAGGAACTATGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCAgctagcaccaag
ggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaa
ggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctg
tcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctac
atctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagaaagttgagcccaaatcttgtgacaaaactca
cacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggaca
ccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttc
aactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccg
tgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaag
ccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgccc
ccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgc
cgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctcct
tcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcat
gaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga
The heavy chain amino acid sequence (470a.a.) that anti-PTPRS is fitted together to 13G5-57 antibody is shown in following (SEQ ID NO：
96).Capitalization shows chimeric 13G5-57VH variable regions, and lowercase shows human IgG1's heavy chain constant region.
MNLGLSLIFLVLVLKGVQCEVKLVESGGGLVQPGGSLKLSCATSGFTFSDYYMYWVRQTPEKRLEWVAYISNGGGST
YYPDTVKGRFTISRDNAKNTLYLQMSRLKSEDTAMYYCARHVYYGRNYAMDYWGQGTSVTVSSastkgpsvfplaps
skstsggtaalgclvkdyfpepvtvswnsgaltsgvhtfpavlqssglyslssvvtvpssslgtqtyicnvnhkpsn
tkvdkkvepkscdkthtcppcpapellggpsvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevh
naktkpreeqynstyrvvsvltvlhqdwlngkeykckvsnkalpapiektiskakgqprepqvytlppsrdeltknq
vsltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqk
slslspgk
The light chain nucleic acid sequence (705bp) that anti-PTPRS is fitted together to 13G5-57 antibody is shown in following (SEQ ID NO：97).Greatly
Female display of writing is fitted together to 13G5-57VL variable regions, and lowercase shows people's Ig κ constant region of light chain.
ATGATGTCCTCTGCTCAGTTCCTTGGTCTCCTGTTGCTCTGTTTTCAAGGTACCAGATGTGATATCCAGATGACACA
GACTACATCCTCCCTGTCTGCCTCTCTGGGAGACAGAGTCACCATCAGTTGCAGGGCAAGTCAGGACATTAGCAATT
ATTTAAACTGGTATCAGCAGAAACCAGATGGAACTGTTAAACTCCTGATCTACTACACATCAAGATTACACTCAGGA
GTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGAACAGATTATTCTCTCACCATTAGCAACCTGGAGCAAGAAGATAT
TGCCACTTACTTTTGCCAACAGGGTAATACGCTTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAAcgaa
ctgtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgc
ctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactccca
ggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagact
acgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacagg
ggagagtgctag
The amino acid sequence (234a.a.) that anti-PTPRS is fitted together to the light chain of 13G5-57 antibody is shown in following (SEQ ID NO：
98).Capitalization shows chimeric 13G5-57VL variable regions, and lowercase shows people's Ig κ constant region of light chain.
MMSSAQFLGLLLLCFQGTRCDIQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVKLLIYYTSRLHSG
VPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEIKrtvaapsvfi
fppsdeqlksgtasvvcllnnfypreakvqwkvdnalqsgnsqesvteqdskdstyslsstltlskadyekhkvyac
evthqglsspvtksfnrgec
The heavy chain of the chimeric 22H8-84 antibody prepared and the nucleotide sequence and amino acid sequence of light chain are respectively with one sequence
Numbering represents.
The heavy chain nucleic acid sequence (1413bp) that anti-PTPRS is fitted together to 22H8-84 antibody is shown in following (SEQ ID NO：99).
Capitalization shows chimeric 22H8-84VH variable regions, and lowercase shows human IgG1's heavy chain constant region.
ATGGAATGTAACTGGATACTTCCTTTTATTCTGTCAGTAACTTCAGGTGTCTACTCACAGGTTCAGCTCCAGCAGTC
TGGGGCTGAGCTGGCAAGACCTGGGGCTTCAGTGAAGTTGTCCTGCAAGGCTTCTGGCTACACCTTTACTAGCTACT
GGATGCAGTGGGTAAAACAGAGGCCTGGACAGGGTCTGGAATGGATTGGGGCTATTTATCCTGGAGATGGTGATACT
AGGTACACTCAGAAGTTCAAGGGCAAGGCCACATTGACTGCAGATAAATCCTCCAGCACAGCCTACATGCAACTCAG
CAGCTTGGCATCTGAGGACTCTGCGGTCTATTACTGTGCAAGAAGGATTTACTACGGCTATTACTATGCTATGGACT
ACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCAgctagcaccaagggcccatcggtcttccccctggcaccctcc
tccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtc
gtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctca
gcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaac
accaaggtggacaagaaagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaact
cctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtca
catgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcat
aatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcacca
ggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatct
ccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccag
gtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccgga
gaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggaca
agagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaag
agcctctccctgtctccgggtaaatga
The heavy chain amino acid sequence (470a.a.) that anti-PTPRS is fitted together to 22H8-84 antibody is shown in following (SEQ ID NO：
100).Capitalization shows chimeric 22H8-84VH variable regions, and lowercase shows human IgG1's heavy chain constant region.
MECNWILPFILSVTSGVYSQVQLQQSGAELARPGASVKLSCKASGYTFTSYWMQWVKQRPGQGLEWIGAIYPGDGDT
RYTQKFKGKATLTADKSSSTAYMQLSSLASEDSAVYYCARRIYYGYYYAMDYWGQGTSVTVSSastkgpsvfplaps
skstsggtaalgclvkdyfpepvtvswnsgaltsgvhtfpavlqssglyslssvvtvpssslgtqtyicnvnhkpsn
tkvdkkvepkscdkthtcppcpapellggpsvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevh
naktkpreeqynstyrvvsvltvlhqdwlngkeykckvsnkalpapiektiskakgqprepqvytlppsrdeltknq
vsltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqk
slslspgk
The light chain nucleic acid sequence (717bp) that anti-PTPRS is fitted together to 22H8-84 antibody is shown in following (SEQ ID NO：101).
Capitalization shows chimeric 22H8-84VL variable regions, and lowercase shows people's Ig κ constant region of light chain.
ATGGAGACAGACACAATCCTGCTATGGGTGCTGCTGCTCTGGGTTCCAGGCTCCACTGGTGACATTGTGCTGACCCA
ATCTCCAGCTTCTTTGGCTGTGTCTCTAGGGCAGAGGGCCACCATCTCCTGCAAGGCCAGCCAAAGTGTTGATTATG
ATGGTGATAGTTATATGAACTGGTACCAACAGAAACCAGGACAGCCACCCAAACTCCTCATCTATGCTGCATCCAAT
CTAGAATCTGGGATCCCAGCCAGGTTTAGTGGCAGTGGGTCTGGGACAGACTTCACCCTCAACATCCATCCTGTGGA
GGAGGAGGATGCTGCAACCTATTACTGTCAGCAAAGTAATGAGGATCCTCTCACGTTCGGTGCTGGGACCAAGCTGG
AGCTGAAAcgaactgtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcc
tctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatc
gggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctga
gcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaag
agcttcaacaggggagagtgctag
The light-chain amino acid sequence (238a.a.) that anti-PTPRS is fitted together to 22H8-84 antibody is shown in following (SEQ ID NO：
102).Capitalization shows chimeric 22H8-84VL variable regions, and lowercase shows people's Ig κ constant region of light chain.
METDTILLWVLLLWVPGSTGDIVLTQSPASLAVSLGQRATISCKASQSVDYDGDSYMNWYQQKPGQPPKLLIYAASN
LESGIPARFSGSGSGTDFTLNIHPVEEEDAATYYCQQSNEDPLTFGAGTKLELKrtvaapsvfifppsdeqlksgta
svvcllnnfypreakvqwkvdnalqsgnsqesvteqdskdstyslsstltlskadyekhkvyacevthqglsspvtk
sfnrgec
The heavy chain of the chimeric 49F2-30 antibody prepared and the nucleotide sequence and amino acid sequence of light chain are respectively with one sequence
Numbering represents.
The nucleotide sequence (1413bp) that anti-PTPRS is fitted together to the heavy chain of 49F2-30 antibody is shown in following (SEQ ID NO：
35).Capitalization shows chimeric 49F2-30VH variable regions, and lowercase shows human IgG1's heavy chain constant region.
ATGAACTTCGGGCTCAGGTTGATTTTCCTTGCCCTCATTTTAAAAGGTGTCCAGTGTGAGGTGCAGCTGGTGGAGTC
TGGGGGAGACTTAGTGAAGCCTGGAGGGTCCCTGAAACTCTCCTGTGCAGCCTCTGGATTCATTTTCAGTAGCTATG
GCATGTCTTGGGTTCGCCAGACTCCAGACAAGAGGCTGGAGTGGGTCGCAACCATTAGTAGTGGTGGTAGTGACACC
TATTATCCAGACAGTGTGAAGGGGCGATTCACCATCTCCAGAGACAATGCCAACAACACCCTGTACCTGCAAATGAG
CAGTCTGAAGTCTGAGGACACAGCCATGTATTACTGTGCAAGACAGGTCTACTATGGTCTTTACTGGTATTTCGATG
TCTGGGGCGCAGGGACCACGGTCACCGTCTCCTCAgctagcaccaagggcccatcggtcttccccctggcaccctcc
tccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtc
gtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctca
gcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaac
accaaggtggacaagaaagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaact
cctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtca
catgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcat
aatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcacca
ggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatct
ccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccag
gtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccgga
gaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggaca
agagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaag
agcctctccctgtctccgggtaaatga
The amino acid sequence (470a.a.) that anti-PTPRS is fitted together to the heavy chain of 49F2-30 antibody is shown in following (SEQ ID NO：
36).Capitalization shows chimeric 49F2-30VH variable regions, and lowercase shows human IgG1's heavy chain constant region.
MNFGLRLIFLALILKGVQCEVQLVESGGDLVKPGGSLKLSCAASGFIFSSYGMSWVRQTPDKRLEWVATISSGGSDT
YYPDSVKGRFTISRDNANNTLYLQMSSLKSEDTAMYYCARQVYYGLYWYFDVWGAGTTVTVSSastkgpsvfplaps
skstsggtaalgclvkdyfpepvtvswnsgaltsgvhtfpavlqssglyslssvvtvpssslgtqtyicnvnhkpsn
tkvdkkvepkscdkthtcppcpapellggpsvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevh
naktkpreeqynstyrvvsvltvlhqdwlngkeykckvsnkalpapiektiskakgqprepqvytlppsrdeltknq
vsltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqk
slslspgk
The nucleotide sequence (705bp) that anti-PTPRS is fitted together to the light chain of 49F2-30 antibody is shown in following (SEQ ID NO：37).
Capitalization shows chimeric 49F2-30VL variable regions, and lowercase shows people's Ig κ constant region of light chain.
ATGGAGTCACAGATTCAGGTCTTTGTATTCGTGTTTCTCTGGTTGTCTGGTGTTGACGGAGACATTGTGATGACCCA
GTCTCACAAATTCATGTCCACATCAGTAGGAGACAGGGTCAGCATCATTTGTAAGGCCAGTCAGGATGTGAATACTG
CTGTAGCCTGGTATCAACAGAAACCAGGACAATCTCCTAAATTACTGATTTACTCGGCATCCTACCGGTACACTGGA
GTCCCTGATCGCTTCACTGGCAGTGGATCTGGGACGGATTTCACTTTCACCATCAGCAGTGTGCAGGCTGAAGACCT
GGCAATTTATTACTGTCAGCAACATTATAGTACTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAAcgaa
ctgtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgc
ctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactccca
ggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagact
acgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacagg
ggagagtgctag
The amino acid sequence (234a.a.) that anti-PTPRS is fitted together to the light chain of 49F2-30 antibody is shown in following (SEQ ID NO：
38).Capitalization shows chimeric 49F2-30VL variable regions, and lowercase shows people's Ig κ constant region of light chain.
MESQIQVFVFVFLWLSGVDGDIVMTQSHKFMSTSVGDRVSIICKASQDVNTAVAWYQQKPGQSPKLLIYSASYRYTG
VPDRFTGSGSGTDFTFTISSVQAEDLAIYYCQQHYSTPYTFGGGTKLEIKrtvaapsvfifppsdeqlksgtasvvc
llnnfypreakvqwkvdnalqsgnsqesvtcqdskdstyslsstltlskadyekhkvyacevthqglsspvtksfnr
gec
Embodiment 6
The Anti-Human PTPRS chimeric antibodies (ch49F2-30, ch9H5-4, ch13G5-57 and ch22H8-84) of preparation resist
Body dependent cellular cytotoxicity
Measure antibody-dependent cellular cytotoxicity (ADCC activity).(taken off by using cytotoxicity from the lactose of cell release
What the measured value of hydrogen enzyme (LDH) calculated) it is used as index to obtain activity.It is pure by centrifugation by specific gravity using HISTOPAQUE-1077
It is turned to the human peripheral blood mononuclear cell of effect daughter cell.CHO (Chinese hamster ovum are utilized as target target cell, having used
Nest cell strain) pressure converted the cell (2x10 of hPTPRS genes4/ hole).Effect daughter cell is mixed with target cell with
Just its ratio becomes 10:1、20:1、40:1 and 80:1, it with the addition of Anti-Human's PTPRS chimeric antibodies of 10 μ g/ml preparation
(ch49F2-30, ch9H5-4, ch13G5-57 and ch22H8-84) or control antibodies Synagis, mixture is incubated 4 at 37 DEG C
Hour to evaluate the cytotoxic effect of antibody.As a result, ch49F2-30, ch9H5-4 of anti-hPTPRS chimeric antibodies,
Ch13G5-57 and ch22H8-84 cracked in a manner of effector cell's number dependence target hPTPRS/CHO cells (Figure 17 A and
Figure 17 B).Cytotoxicity of anti-PTPRS chimeric antibodies prepared by the result display selectively display to expression PTPRS cell.
It has studied effect of the anti-PTPRS antibody to pDC.PBMC is separated from human peripheral, by its Anti-Human with 10 μ g/ml
PTPRS chimeric antibodies mix, and cultivate 24 hours.Then, 24 hours are stimulated to induce IFN α to produce using CpG2216, it is described
CpG2216 is the part for the Toll-like receptor 9 expressed in pDC.24h after CpG is stimulated, test the yield of IFN α.As a result, IFN α
The place of Anti-Human PTPRS chimeric antibodies (ch49F2-30, ch9H5-4, ch13G5-57 and ch22H8-84) that is produced of generation
Reason complete inhibition (Figure 18 A).
In addition, when ch49F2-30, ch9H5-4, ch13G5-57 and ch22H8-84 processing after 6 it is small when collect cell and
When confirming pDC1 by using double dyeing of anti-BDCA2 antibody and anti-BDCA4 antibody, it is found that pDC colonies reduce by more than control
The Synagis processing (Figure 18 B and 18C) of antibody.
Industrial usability
Exempt from the invention provides specific recognition people PTPRS (immunogene for being used to produce antibody) antibody and for utilizing
The method that epidemic focus produces Anti-Human's PTPRS antibody.
Accession number
FERM BP-11356
FERM BP-11357
FERM BP-11358
FERM BP-11359
FERM BP-11360
FERM BP-11361
FERM BP-11362
FERM BP-11363
The text of unordered list
SEQ ID NO：3：Forward primer
SEQ ID NO：4：Reverse primer
SEQ ID NO：5：Forward primer
SEQ ID NO：6：Reverse primer
SEQ ID NO：7：Forward primer
SEQ ID NO：8：Reverse primer
SEQ ID NO：9：Forward primer
SEQ ID NO：10：Reverse primer
SEQ ID NO：11：Antisense primer
SEQ ID NO：12：Anchor primer
SEQ ID NO：12：N is deoxyinosine.
SEQ ID NO：13：Antisense primer
SEQ ID NO：14：AUAP primers
SEQ ID NO：15：Antisense primer
SEQ ID NO：16：Antisense primer
SEQ ID NO：17：Primer
SEQ ID NO：18：Primer
SEQ ID NO：19：Primer
SEQ ID NO：20：Primer
SEQ ID NO：21：Primer
SEQ ID NO：22：Primer
SEQ ID NO：23：Primer
SEQ ID NO：24：Primer
SEQ ID NO：35：Anti- PTPRS is fitted together to the heavy chain nucleic acid sequence of 49F2-30 antibody
SEQ ID NO：36：Anti- PTPRS is fitted together to the heavy chain amino acid sequence of 49F2-30 antibody
SEQ ID NO：37：Anti- PTPRS is fitted together to the light chain nucleic acid sequence of 49F2-30 antibody
SEQ ID NO：38：Anti- PTPRS is fitted together to the light-chain amino acid sequence of 49F2-30 antibody
SEQ ID NO：39：Primer
SEQ ID NO：40：Primer
SEQ ID NO：41：Primer
SEQ ID NO：42：Primer
SEQ ID NO：73：Forward primer
SEQ ID NO：74：Reverse primer
SEQ ID NO：75：Forward primer
SEQ ID NO：76：Reverse primer
SEQ ID NO：77：Forward primer
SEQ ID NO：78：Reverse primer
SEQ ID NO：79：Forward primer
SEQ ID NO：80：Reverse primer
SEQ ID NO：81：Forward primer
SEQ ID NO：82：Reverse primer
SEQ ID NO：83：Forward primer
SEQ ID NO：84：Reverse primer
SEQ ID NO：85：Forward primer
SEQ ID NO：86：Reverse primer
SEQ ID NO：87：Forward primer
SEQ ID NO：88：Reverse primer
SEQ ID NO：89：Forward primer
SEQ ID NO：90：Reverse primer
SEQ ID NO：91：Anti- PTPRS is fitted together to the heavy chain nucleic acid sequence of 9H5-4 antibody
SEQ ID NO：92：Anti- PTPRS is fitted together to the heavy chain amino acid sequence of 9H5-4 antibody
SEQ ID NO：93：Anti- PTPRS is fitted together to the light chain nucleic acid sequence of 9H5-4 antibody
SEQ ID NO：94：Anti- PTPRS is fitted together to the light-chain amino acid sequence of 9H5-4 antibody
SEQ ID NO：95：Anti- PTPRS is fitted together to the heavy chain nucleic acid sequence of 13G5-57 antibody
SEQ ID NO：96：Anti- PTPRS is fitted together to the heavy chain amino acid sequence of 13G5-57 antibody
SEQ ID NO：97：Anti- PTPRS is fitted together to the light chain nucleic acid sequence of 13G5-57 antibody
SEQ ID NO：98：Anti- PTPRS is fitted together to the light-chain amino acid sequence of 13G5-57 antibody
SEQ ID NO：99：Anti- PTPRS is fitted together to the heavy chain nucleic acid sequence of 22H8-84 antibody
SEQ ID NO：100：Anti- PTPRS is fitted together to the heavy chain amino acid sequence of 22H8-84 antibody
SEQ ID NO：101：Anti- PTPRS is fitted together to the light chain nucleic acid sequence of 22H8-84 antibody
SEQ ID NO：102：Anti- PTPRS is fitted together to the light-chain amino acid sequence of 22H8-84 antibody
Claims (20)
1. a kind of monoclonal antibody or the fragment including its antigen binding domain, the antibody or fragment combination plasmacytoid dendritic are thin
The extracellular domain of people's acceptor type protein tyrosine phosphatase σ (people PTPRS) on born of the same parents, and comprising：
(A)SEQ ID NO:Heavy chain CDR1, SEQ ID NO shown in 45:Heavy chain CDR2, SEQ ID NO shown in 46:Shown in 47
Heavy chain CDR3, SEQ ID NO:Light chain CDR1, SEQ ID NO shown in 50:Light chain CDR2 and SEQ ID NO shown in 51:
It is at least one in light chain CDR3 shown in 52；
(B)SEQ ID NO:Heavy chain CDR1, SEQ ID NO shown in 55:Heavy chain CDR2, SEQ ID NO shown in 56:Shown in 57
Heavy chain CDR3, SEQ ID NO:Light chain CDR1, SEQ ID NO shown in 60:Light chain CDR2 and SEQ ID NO shown in 61:
It is at least one in light chain CDR3 shown in 62；
(C)SEQ ID NO:Heavy chain CDR1, SEQ ID NO shown in 65:Heavy chain CDR2, SEQ ID NO shown in 66:Shown in 67
Heavy chain CDR3, SEQ ID NO:Light chain CDR1, SEQ ID NO shown in 70:Light chain CDR2 and SEQ ID NO shown in 71:
It is at least one in light chain CDR3 shown in 72；
(D)SEQ ID NO:Heavy chain CDR1, SEQ ID NO shown in 27:Heavy chain CDR2, SEQ ID NO shown in 28:Shown in 29
Heavy chain CDR3, SEQ ID NO:Light chain CDR1, SEQ ID NO shown in 32:Light chain CDR2 and SEQ ID NO shown in 33:
It is at least one in light chain CDR3 shown in 34；
(E)SEQ ID NO:Weight chain variable district and/or SEQ ID NO shown in 44:Light chain variable district shown in 49；
(F)SEQ ID NO:Weight chain variable district and/or SEQ ID NO shown in 54:Light chain variable district shown in 59；
(G)SEQ ID NO:Weight chain variable district and/or SEQ ID NO shown in 64:Light chain variable district shown in 69；Or
(H)SEQ ID NO:Weight chain variable district and/or SEQ ID NO shown in 26:Light chain variable district shown in 31.
2. the monoclonal antibody or fragment of claim 1, wherein the antibody is by with accession number FERM BP-11356 preservations
Hybridoma 9H5-4, with the hybridoma 10F7-38 of accession number FERM BP-11357 preservations, with accession number FERM BP-11358 protect
The hybridoma 13G5-52 of Tibetan, with the hybridoma 13G5-57 of accession number FERM BP-11359 preservations, with accession number FERM BP-
The hybridoma 14A8-85 of 11360 preservations, with the hybridoma 22H8-84 of accession number FERM BP-11361 preservations or with accession number
Caused by the hybridoma 49F2-30 of FERM BP-11362 preservations.
3. the monoclonal antibody or fragment of claim 1, it is chimeric or humanization.
4. the monoclonal antibody or fragment of claim 3, wherein including：
(A)SEQ ID NO:Heavy chain and/or SEQ ID NO shown in 92:Light chain shown in 94；
(B)SEQ ID NO:Heavy chain and/or SEQ ID NO shown in 96:Light chain shown in 98；
(C)SEQ ID NO:Heavy chain and/or SEQ ID NO shown in 100:Light chain shown in 102；Or
(D)SEQ ID NO:Heavy chain and/or SEQ ID NO shown in 36:Light chain shown in 38.
A kind of 5. hybridoma for the monoclonal antibody for producing claim 1.
6. a kind of monoclonal antibody or the fragment including its antigen binding domain, the antibody or fragment combination plasmacytoid dendritic are thin
The extracellular domain of people's acceptor type protein tyrosine phosphatase σ (people PTPRS) on born of the same parents, the antibody is to pass through genetic recombination
From the Antibody preparation as caused by with the hybridoma 55E7-79 of accession number FERM BP-11363 preservations.
7. the monoclonal antibody or fragment of claim 6, the antibody has been transplanted by with accession number FERM BP-11363 preservations
The CDR of antibody caused by hybridoma 55E7-79.
8. the monoclonal antibody or fragment of claim 6, wherein the antibody is chimeric or humanization.
9. a kind of monoclonal antibody or the fragment including its antigen binding domain, the antibody or fragment combination plasmacytoid dendritic are thin
The extracellular domain of people's acceptor type protein tyrosine phosphatase σ (people PTPRS) on born of the same parents, and comprising by with accession number
The CDR of antibody caused by the hybridoma 55E7-79 of FERM BP-11363 preservations.
10. the monoclonal antibody or fragment of claim 9, wherein the antibody is chimeric or humanization.
11. the monoclonal antibody or fragment of claim 9, wherein the antibody is by with accession number FERM BP-11363 preservations
Hybridoma 55E7-79 caused by.
12. one kind is used to produce celliferous method, the cell produces the people's acceptor type combined on plasmacytoid dendritic cells
The monoclonal antibody of Protein-tyrosine-phosphatase σ (people PTPRS) extracellular domain, methods described include：
1) cell for the exogenous proteins for expressing the extracellular domain for including people PTPRS is applied to the animal for treating immunity inoculation,
With
2) antibody-producing cell of the monoclonal antibody is produced from the animal selection through immunity inoculation.
13. a kind of monoclonal antibody or the fragment including its antigen binding domain, the antibody or fragment combination plasmacytoid dendritic
The extracellular domain of people's acceptor type protein tyrosine phosphatase σ (people PTPRS) on cell, wherein the monoclonal antibody
It can be obtained from by cell caused by the method described in claim 12.
14. a kind of monoclonal antibody or the fragment including its antigen binding domain, the antibody or fragment combination plasmacytoid dendritic
The extracellular domain of people's acceptor type protein tyrosine phosphatase σ (people PTPRS) on cell, and suppress in the cell
IFN generation and/or the survival of cell, wherein the antibody can obtain through the following steps：
1) the thin of the protein for the extracellular domain for including people PTPRS is expressed to the animal administration of exogenous for treating immunity inoculation
Born of the same parents；
2) the antibody producing of the antibody with reference to people PTPRS is produced from the antibody-producing cell selection of the animal through immunity inoculation
Cell；With
3) antibody-producing cell of selection in (2) is cultivated, and identification people PTPRS is collected, with reference to Plasmacytoid tree from culture
Prominent cell simultaneously suppresses the active antibody of the plasmacytoid dendritic cells；
The activity of wherein described plasmacytoid dendritic cells is IFN generation and/or cell survival.
15. one kind is used for the method for detecting plasmacytoid dendritic cells (pDC), it is included the monoclonal described in claim 13
Antibody or fragment contact with subject cell, and detection is with reference to the monoclonal antibody or fragment of the cell.
16. monoclonal antibody according to claim 13 or fragment are preparing the purposes in being used to detect pDC reagent.
17. a kind of active ex vivo approach for being used to suppress plasmacytoid dendritic cells, it is included claim 1-4,6-
Any one of 11 and 14 monoclonal antibody or fragment contacts with plasmacytoid dendritic cells, wherein the plasmacytoid dendritic is thin
The activity of born of the same parents is IFN generation and/or cell survival.
18. the monoclonal antibody or fragment of any one of claim 1-4,6-11 and 14 are being prepared for suppressing Plasmacytoid
Purposes in the active reagent of dendritic cells, wherein the activity of the plasmacytoid dendritic cells is IFN generation and/or thin
Born of the same parents are survived.
19. the monoclonal antibody or fragment of any one of claim 1-4,6-11 and 14 are being prepared by suppressing Plasmacytoid
Purposes in the medicament that is active and treating disease of dendritic cells, wherein the activity of the plasmacytoid dendritic cells is IFN
Generation and/or cell survival.
20. the monoclonal antibody or fragment of any one of claim 1-4,6-11 and 14 are being prepared for treating autoimmunity
Purposes in the medicament of disease.
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| JP2011101752 | 2011-04-28 | ||
| PCT/JP2012/061795 WO2012148003A1 (en) | 2011-04-28 | 2012-04-27 | ANTI-HUMAN RECEPTOR-TYPE PROTEIN TYROSINE PHOSPHATASE σ ANTIBODY |
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| CN103703372A CN103703372A (en) | 2014-04-02 |
| CN103703372B true CN103703372B (en) | 2018-01-26 |
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| CA2691378A1 (en) | 2007-07-02 | 2009-01-08 | Oncomed Pharmaceuticals, Inc. | Antibody against human r-spondin (rspo) and use thereof for inhibition of beta-catenin signaling and treatment of cancer |
| CA2834243C (en) * | 2011-04-28 | 2019-08-27 | Sbi Biotech Co., Ltd. | Anti-human receptor-type protein tyrosine phosphatase .sigma. antibody |
| WO2013012747A1 (en) | 2011-07-15 | 2013-01-24 | Oncomed Pharmaceuticals, Inc. | Rspo binding agents and uses thereof |
| JP6335896B2 (en) | 2012-07-13 | 2018-05-30 | オンコメッド ファーマシューティカルズ インコーポレイテッド | RSPO3 binding agent and method of use thereof |
| DK2877494T3 (en) | 2012-07-23 | 2020-09-21 | La Jolla Inst Allergy & Immunology | PTPRS and proteoglycans in autoimmune disease |
| US10023653B2 (en) * | 2014-02-04 | 2018-07-17 | Monash University | Anti-metalloprotease antibody for diagnosis and treatment of cancers |
| US11160873B2 (en) * | 2014-02-04 | 2021-11-02 | Monash University | Anti-metalloprotease antibody for diagnosis and treatment of cancers |
| US10064937B2 (en) | 2014-09-16 | 2018-09-04 | Oncomed Pharmaceuticals, Inc. | Treatment of dermal fibrosis |
| BR112017011170A2 (en) * | 2014-12-18 | 2018-02-27 | Hoffmann La Roche | method for determining the complement-dependent cytotoxicity of a composition |
| EA201992248A1 (en) * | 2017-03-23 | 2020-02-06 | Дзе Трастиз Оф Дзе Юниверсити Оф Пенсильвания | ANTI-C5a-ANTIBODIES AND THEIR APPLICATION |
| WO2018176019A1 (en) * | 2017-03-24 | 2018-09-27 | The Regents Of The University Of California | Proteoglycan irregularities in abnormal fibroblasts and therapies based therefrom |
| WO2019028182A2 (en) * | 2017-08-01 | 2019-02-07 | Remd Biotherapeutics, Inc. | Cancer treatment using antibodies that bind human cd134 (ox40) receptor |
| WO2020023679A1 (en) * | 2018-07-25 | 2020-01-30 | Accurus Biosciences, Inc. | Novel cldn 18.2-specific monoclonal antibodies and methods of use thereof |
| MX2022004058A (en) * | 2019-10-04 | 2022-07-13 | Albert Einstein College Of Medicine | Kir3dl3 is an inhibitory receptor of the immune system and uses thereof. |
| CN114426582B (en) * | 2022-01-07 | 2022-11-25 | 陕西脉元生物科技有限公司 | Nerve specific enolase monoclonal antibody and preparation method and application thereof |
| CN116041526B (en) * | 2022-11-14 | 2023-09-29 | 华中农业大学 | Mouse anti-grass carp IgT monoclonal antibody, preparation method and application thereof |
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| WO1995009656A1 (en) | 1993-10-01 | 1995-04-13 | New York University | Novel receptor-type phosphotyrosine phosphatase-sigma |
| JP4498136B2 (en) | 2002-08-01 | 2010-07-07 | Ｓｂｉバイオテック株式会社 | Method for detecting mouse interferon-producing cells |
| RU2312109C2 (en) * | 2003-03-14 | 2007-12-10 | Тайсо Фармасьютикал Ко., Лтд. | Monoclonal antibody and hybridoma producing its |
| WO2005102456A1 (en) | 2004-03-27 | 2005-11-03 | The Arizona Board Of Regents On Behalf Of The University Of Arizona | Composition and method for cancer treatment |
| US20080008719A1 (en) | 2004-07-10 | 2008-01-10 | Bowdish Katherine S | Methods and compositions for the treatment of prostate cancer |
| JP2009510102A (en) | 2005-09-29 | 2009-03-12 | ヴァイラル ロジック システムズ テクノロジー コーポレーション | Immunomodulatory compositions and uses thereof |
| WO2009003274A1 (en) | 2007-06-29 | 2009-01-08 | The Hospital For Sick Children | Susceptibility gene for inflammatory bowel disease |
| EP2356446A4 (en) * | 2008-11-14 | 2014-03-19 | Brigham & Womens Hospital | Therapeutic and diagnostic methods relating to cancer stem cells |
| US20120231014A1 (en) * | 2009-08-18 | 2012-09-13 | Case Western Reserve University | Neural Regeneration |
| WO2011097327A2 (en) * | 2010-02-02 | 2011-08-11 | La Jolla Institute For Allergy & Immunology | Compositions and methods of modulating receptor protein tyrosine phosphatases |
| CA2834243C (en) * | 2011-04-28 | 2019-08-27 | Sbi Biotech Co., Ltd. | Anti-human receptor-type protein tyrosine phosphatase .sigma. antibody |
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